This work was supported by grants provided by the National Institutes of Health (NIH), R01 CA200669 to JF and R01 CA121107 to GRJT, and by a postdoctoral fellowship grant from Susan G. Komen for the Cure to IK (PDF12229484).

1. Synthesis of Aspirin-fumarate Prodrug GTCpFE
<ol>
	<li>Using a plastic plunger syringe, measure 0.81 ml (20 mmol) of methanol and mix it in water (10 ml) in a round bottom flask. Cool the resulting mixture to 0 &#176;C by placing the flask in an ice-water bath. Add 4-hydroxybenzyl alcohol (2.48 mg, 20 mmol) and stir the reaction mixture until the solution is clear.</li>
	<li>Prepare a solution of O-acetylsalicyloyl chloride (3.77 mg, 19 mmol) in anhydrous toluene (10 ml) by weighing the desired amount of O-acetylsalicyloyl chloride and dissolving it in the solvent in a separate flask. Using a plastic plunger syringe, add this solution to the mixture prepared in step 1.1, and leave the reaction stirring at 0 &#176;C.</li>
	<li>Monitor the reaction using thin layer chromatography (TLC). The TLC plates should have silica as a stationary phase.
	<ol>
		<li>Prepare a mobile phase composed of 20:80 ethyl acetate (AcOEt)/hexane. In a TLC chamber (or small container) add 5 ml of mobile phase to cover the bottom of the chamber. 
		NOTE: The amount of mobile phase depends on the chamber selected. Always add enough to cover the bottom, but once the TLC is placed inside, the solvent line should not surpass the height where the compounds are spotted.</li>
		<li>Take a sample of the reaction with a syringe (0.2 ml), place it in a small vial, and dilute it with ethyl acetate (2-3 drops). With a TLC spotter, carefully take a sample of the diluted reaction mixture and spot it in the TLC. 
		NOTE: The spots should be 1-2 mm and this should be done at the lower 1/4 inch of the TLC plate.</li>
		<li>On the same TLC, place a spot of a solution of O-acetylsalicyloyl chloride (0.2 mg in 0.5 ml of ethyl acetate) as a comparison. Once the spots are dry, place it in the chamber and let it run until the solvent front has almost reached the top of the TLC.</li>
		<li>Take the TLC out, let it dry and visualize it under a UV lamp. The reaction is complete when the O-acetylsalicyloyl chloride spot disappears from the reaction mixture.</li>
		<li>When the reaction is completed, remove the ice-water bath, and allow the reaction to stir at room temperature for 20 hr.</li>
	</ol>
	</li>
	<li>Filter the precipitate using a Buchner funnel with a fritted disk of medium porosity. Place the solid in a scintillation vial, and leave the compound in vacuo overnight in a desiccator at room temperature. Use phosphorus pentoxide (P2O5) as the desiccant.</li>
	<li>Dry a round bottom flask by placing it in an oven at 80 &#176;C for at least two days before setting up this reaction. Alternatively, seal the flask with a septum and pierce it with a needle that is connected to a vacuum pump system. Turn the vacuum pump on and dry the flask using a heat gun. Allow to cool, and repeat this step 2 more times.</li>
	<li>Inflate a balloon using argon gas, and connect it to a plastic syringe (whose plunger has been removed) with a needle. Turn off the vacuum pump and remove the needle attached to it that was inserted in the now dried round bottom flask. Insert the needle connected to the argon balloon through the septum sealing the round bottom flask.
	<ol>
		<li>Add 4-hydroxymethylphenol ester of 2-acetyloxybenzoic acid (100 mg, 0.349 mmol), 4-dimethylaminopyridine (4 mg, 0.033 mmol) and triethylamine (53 mg, 0.523 mmol) to a separate flask, then dissolved them in anhydrous tetrahydrofuran (5 ml). With a plastic syringe attached to a needle, add this solution to the sealed round bottom flask. Cool the mixture down to 0 &#176;C by placing the flask in an ice water bath.</li>
	</ol>
	</li>
	<li>To the previous mixture, add a solution of ethyl fumaroyl chloride (68 mg, 0.419 mmol) in anhydrous tetrahydrofuran (5 ml), drop-wise over a period of 10 min. Stir the resulting solution at 0-5 &#176;C for 2-4 hr.</li>
	<li>Extract the reaction mixture with ethyl acetate (100 ml) and brine (50 ml). 
	NOTE: Brine is a saturated solution of sodium chloride (NaCl). To prepare it, place 200 ml of water in a clean glass container then start adding NaCl (while stirring) until it doesn&#39;t dissolve anymore.
	<ol>
		<li>After extraction, remove the aqueous phase and repeat the latter step two more times.</li>
		<li>Dry the mixture by adding sodium sulfate to the organic phase, until the solid does not clump up when the glassware is stirred. Using another Buchner funnel with a fritted disk, filter the mixture to a round bottom flask to remove the sodium sulfate, then evaporate to dryness using a rotary evaporator with the temperature set at 40 &#176;C.</li>
	</ol>
	</li>
	<li>Using a TLC, determine a proper mobile phase to use in column chromatography. 
	NOTE: For this experiment a gradient of 20:80 AcOEt/hexane was used.</li>
	<li>Prepare a column with silica gel and the appropriate solvent phase. Once the compound has been added, add a layer of sodium sulfate (or sand) to protect the column as the solvent is added.
	<ol>
		<li>As the column runs, monitor the eluate by TLC. Spot every other test tube as they are collected from the column. When the product of interest elutes, mix all samples containing pure product into a large round bottom flask, and dry them using a rotary evaporator with the bath temperature set at 40 &#176;C. 
		NOTE: Product should appear as a white solid.</li>
	</ol>
	</li>
	<li>To confirm the structure of GTCpFE, collect proton and carbon (1H and 13C, respectively) nuclear magnetic resonance (NMR) spectra as per manufacturer&#39;s instructions. 
	NOTE: In this study a 400 MHz FT NMR spectrometer was used to collect the 1H and 13C spectra. Run at least 25 scans at room temperature to obtain enough data resolution. The NMR peaks observed were the following, 1H NMR (CDCl3): &#948; 1.29 (t, 3 H, 3J = 7.0 Hz, CH3); 2.31 (s, 3 H, CH3); 4.26 (q, 2 H, 3J = 7.0 Hz, COOCH2); 5.25 (s, 2 H, OCH2); 6.89 (s, 2 H, HC=CH); 7.19 (m, 3 H, Ar); 7.42 (m, 3 H, Ar); 7.65 (m, 1 H, Ar); 8.22 (dd, 1 H, 3J = 7.8, 4J = 1.2 Hz, Ar). 13C NMR (CDCl3) &#948;: 169.70, 164.86, 164.75, 162.89, 151.28, 150.69, 134.76, 134.32, 133.26, 133.16, 132.25, 129.81, 126.26, 124.11, 122.47, 122.04, 66.40, 61.43, 21.05, 14.15.</li>
	<li>In addition, collect a high resolution mass spectrum using liquid chromatography mass spectrometry in combination with ion trap and time-of flight technology (LCMS-IT-TOF) for accurate mass measurements as per manufacturer&#39;s instructions. 
	NOTE: In this study (M+NH4+) calculated: 430.1496; observed: 430.1477.</li>
</ol>
2. GTCPFE Inhibits the NF&#312;B Activity in Breast Cancer Cells
<ol>
	<li>Cell Culture Conditions
	<ol>
		<li>Maintain human breast cancer cell lines, MCF-7 and MDA-MB-231 as per standard cell culture techniques and propagate in a humidified incubator at 37 &#176;C with 5% CO2.
		<ol>
			<li>Prepare MCF-7 cell medium from Roswell Park Memorial Institute (RPMI) 1640 medium with phenol red supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, 2 mM L-glutamine, 1% antibiotics penicillin-streptomycin, and 6 ng/ml insulin. Prepare MDA-MB-231 cell medium from Improved Minimum Essential Medium (IMEM) supplemented with 5% FBS, 1% non-essential amino acids, 2 mM L-glutamine, and 1% antibiotics penicillin-streptomycin.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>NF&#954;B-RE Luciferase Reporter Assay
	<ol>
		<li>Trypsinize breast cancer MCF-7 cells using 0.25% trypsin for 5 min at 37 &#176;C, manually count using a hemocytometer and seed in 24-well plates at 90,000 cells per well density.</li>
		<li>The following day, co-transfect cells with plasmid DNA of an NF&#954;B response element (NF&#954;B-RE) luciferase construct, 1 &#181;g/well, along with the promoterless Renilla luciferase construct, 0.2 &#181;g/well. Perform transfections for each treatment group in triplicate.
		<ol>
			<li>Wash cells twice with PBS, and incubate with mixtures of plasmid DNA and 1 &#181;l per well of transfection reagent (e.g. lipofectamine) in serum free media. After 6 hr, change the medium to 1 ml of regular medium (without the antibiotics penicillin and streptomycin).</li>
		</ol>
		</li>
		<li>After 16 hr, add 1 &#181;l of vehicle or different stock solutions of the GTCpFE or ASA drugs to each well. Dissolve drug stock solutions (100, 50, 20, 10 and 1 mM) in dimethyl sulfoxide at 1,000x concentration. After adding the drugs, incubate cells for 2 hr at 37 &#176;C.</li>
		<li>To activate the NF&#954;B pathway, add the pro-inflammatory cytokine TNF&#945; (10 &#181;g/ml stock solution) into each well for a final concentration of 10 ng/ml and incubate for 4 hr. Include a TNF&#945; alone control. Aspirate the medium and store the cells at -80 &#176;C.</li>
		<li>Measure luciferase using a luciferase reporter assay system according to manufacturer&#39;s instructions. 
		NOTE: When using the luciferase assay system (e.g. Dual Luciferase assay system) for the first time, prepare a solution of luciferase assay reagent by re-suspending it in 10 ml of buffer and storing it at -80 &#176;C in 1 ml aliquots.
		<ol>
			<li>Prepare 1x lysis buffer by diluting 5x stock buffer with water. Lyse cells in each well using 100 &#181;l 1x lysis buffer. Incubate the plates on an orbital shaker for 15 min, at medium speed.</li>
			<li>Label one 1.5 ml tube per sample. Thaw luciferase assay reagent to room temperature (50 &#181;l per sample will be needed) and keep in foil. Prepare 1x quenching reagent in a glass vial, 1:49 dilution in buffer and vortex it (50 &#181;l per sample will be needed).</li>
			<li>Add 10 &#181;l of cell lysate to a labeled microcentrifuge tube. Then add 50 &#181;l of luciferase assay reagent and gently vortex. Immediately after, place the tube in a holder and measure the luminescence via the dual luciferase program in the luminometer. Select and press the following: Run Promega Protocols&#160;&#8594; Dual Glo &#8594; OK.</li>
			<li>Add 50 &#181;l of the quenching reagent to the test tube and gently vortex. Measure the luminescence. Repeat these steps for each sample.</li>
			<li>Analyze data using spreadsheet by normalizing NF&#954;B-RE to the Renilla internal control (NF&#954;B-RE/Renilla). Compare inhibitor data to TNF&#945; only control (TNF&#945; only is set at 100%).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>NF&#954;B-Target Gene Transcription
	<ol>
		<li>Seed MCF-7 cells in 6-well plates at 250,000 cells per well in 2 ml medium volume prepared as described in section 2.1.</li>
		<li>The following day, add 2 &#181;l of different GTCpFE 1,000x stocks (100, 50, 20, 10 and 1 mM) for 2 hr prior to adding TNF&#945; 10ng/ml for another 2 hr. Run every treatment in triplicate. Include vehicle and TNF&#945; alone controls.</li>
		<li>Isolate RNA using the guanidinium thiocyanate-phenol-chloroform extraction method according to the instructions of the manufacturer22. Determine RNA concentration (in diethylpyrocarbonate (DEPC)-treated water) and RNA purity using a spectrophotometer. Keep RNA samples on ice or store at -80 &#176;C. Use only RNase-free barrier tips when handling RNA.</li>
		<li>Reverse transcribe 0.5 &#181;g total RNA using a commercially available kit of Moloney murine leukemia virus (M-MLV) reverse transcriptase. 
		NOTE: The kit includes 5x buffer and dithiothreitol (DTT), together with dNTP mixture, and random hexamers mixture.
		<ol>
			<li>Add 0.5 &#181;g of RNA to 200 units of M-MLV, 0.5 mM dNTPs, 100 ng of random hexamers, 10 mM DTT, 1x buffer and DEPC water to a final 10 &#181;l reaction volume. Carry out the reverse transcriptase reaction using a PCR cycler for 50 min at 37 &#176;C, and then 15 min at 70 &#176;C to heat-inactivate the enzyme.</li>
		</ol>
		</li>
		<li>Dilute the resulting cDNA product to 100 &#181;l with double-distilled water and use 2 &#181;l for each subsequent quantitative polymerase chain reaction (PCR) reaction.
		<ol>
			<li>Mix forward and reverse primers for the gene of interest at 1.25 &#181;M concentration each. Prepare a master mix with 2 &#181;l double distilled water, 1 &#181;l of 1.25 &#181;M primer mix and 5 &#181;l of 2x of the dye to enable detection of the double-stranded DNA. Load the 2 &#181;l of cDNA and 8 &#181;l of master mix into 96-well PCR plates.</li>
		</ol>
		</li>
		<li>Carry out quantitative PCR using a real time PCR system (40 cycles, 95-60 &#176;C) according to manufacturer&#39;s instructions and manually analyze the data. 
		NOTE: All PCR primers used have been validated and reported previously23.</li>
		<li>Calculate gene expression fold change using spreadsheet via the &#916;&#916;Ct method, with ribosomal protein 36B4 mRNA serving as the internal control23.</li>
	</ol>
	</li>
</ol>
3. GTCpFE Inhibits Breast Cancer Stem Cells In Vitro
<ol>
	<li>Mammosphere Formation Assay
	<ol>
		<li>Prepare mammosphere (MS) medium by supplementing Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) phenol red-free medium with 1% methyl cellulose. Allow to dissolve by gentle shaking overnight at 4 &#176;C. Filter sterilize the medium and supplement with B27 1x, 1% penicillin and streptomycin, 5 &#181;g/ml insulin, 1 &#181;g/ml hydrocortisone, and 20 ng/ml recombinant human epidermal growth factor.</li>
		<li>Prepare single cells of MDA-MB-231 cell line by trypsin digestion (trypsin 0.25% for 5 min at 37 &#176;C) of monolayer cultures and filter through mesh sieves. Manually count single dissociated cells and plate in 96-well ultra-low attachment plates at a density of 400 cells/well. The next day, add different concentrations of GTCpFE (e.g., final GTCpFE concentrations: 1, 10, 20, 50 &#181;M) to a final volume is 100 &#181;l. Conduct every treatment in triplicate.</li>
		<li>After 7 days of culture in the incubator, acquire images via an imaging software using an inverted microscope. Manually count the number MS &#62;75 &#181;m in diameter. Compare the drug treatment to vehicle control. 
		NOTE: The diameter of MS &#62;75 &#181;m is determined using the imaging software.</li>
	</ol>
	</li>
	<li>CD44+CD24- CSC Immunophenotype
	<ol>
		<li>Trypsinize MDA-MB-231 cells with 0.25% trypsin for 5 min at 37 &#176;C. Count using a hemocytometer and seed in 10 cm dishes at 3 million cells per dish in 10 ml medium as described in section 2.1. Add vehicle (10 &#181;l dimethyl sulfoxide) or GTCpFE (10 &#181;l of 50 mM stock) to cells for 72 hr.</li>
		<li>For vehicle or GTCpFE treatment groups, trypsinize cells (as in step 3.2.1) and distribute 1 million cells in &#39;Test&#39; or &#39;Control&#39; 5 ml polystyrene tubes containing 2 ml of 1x Hank&#39;s Balanced Salt Solution (HBSS) buffer supplemented with 2% FBS.</li>
		<li>To stain for the surface markers CD44 and CD24, spin cells down and add 20 &#181;l of each conjugated antibody and HBSS + 2% FBS to Test tubes for a final 100 &#181;l overall volume (1:5 dilution). Add 100 &#181;l of HBSS + 2% FBS to Control tubes. Include CD44-APC conjugated antibody and CD24-PE antibody single stain controls or the IgG immunoisotype controls.</li>
		<li>Incubate cells in the dark at 4 &#176;C for 30 min. Spin down cells for 5 min at 400 x g and reconstitute in 200 &#181;l HBSS buffer with 2% FBS. Keep cells on ice in the dark.</li>
		<li>Perform Fluorescence-activated cell sorting (FACS) of live cells using a FACS analyzer instrument according to manufacturer&#39;s instructions. Collect at least 50,000 events for each tube. Run treatments in triplicate. 
		NOTE: Gating is based on controls from no staining (Control tube), IgG immunoisotypes, and the CD44-APC and CD24-PE single stains.</li>
		<li>Analyze data using an available flow cytometry software. 
		NOTE: The percent of GTCpFE-treated cell with the CD44+CD24- immunophenotype is estimated and compared to vehicle control.</li>
	</ol>
	</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proc Natl Acad Sci U S A. 100, 3983-3988 (2003).</li><li>Vandermeeren, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethylfumarate+is+an+inhibitor+of+cytokine-induced+nuclear+translocation+of+NF-kappa+B1,+but+not+RelA+in+normal+human+dermal+fibroblast+cells.">Dimethylfumarate is an inhibitor of cytokine-induced nuclear translocation of NF-kappa B1, but not RelA in normal human dermal fibroblast cells.</a> J Invest Dermatol. 116, 124-130 (2001).</li><li>Loewe, R., 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=Dimethylfumarate+inhibits+TNF-induced+nuclear+entry+of+NF-kappa+B/p65+in+human+endothelial+cells.">Dimethylfumarate inhibits TNF-induced nuclear entry of NF-kappa B/p65 in human endothelial cells.</a> J Immunol. 168, 4781-4787 (2002).</li><li>Seidel, 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=Dimethylfumarate+inhibits+NF-{kappa}B+function+at+multiple+levels+to+limit+airway+smooth+muscle+cell+cytokine+secretion.">Dimethylfumarate inhibits NF-{kappa}B function at multiple levels to limit airway smooth muscle cell cytokine secretion.</a> Am J Physiol Lung Cell Mol Physiol. 297, L326-L339 (2009).</li><li>Wilms, 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=Dimethylfumarate+inhibits+microglial+and+astrocytic+inflammation+by+suppressing+the+synthesis+of+nitric+oxide,+IL-1beta,+TNF-alpha+and+IL-6+in+an+in-vitro+model+of+brain+inflammation.">Dimethylfumarate inhibits microglial and astrocytic inflammation by suppressing the synthesis of nitric oxide, IL-1beta, TNF-alpha and IL-6 in an in-vitro model of brain inflammation.</a> J Neuroinflammation. 7, 30 (2010).</li><li>Peng, 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=Dimethyl+fumarate+inhibits+dendritic+cell+maturation+via+nuclear+factor+kappaB+(NF-kappaB)+and+extracellular+signal-regulated+kinase+1+and+2+(ERK1/2)+and+mitogen+stress-activated+kinase+1+(MSK1)+signaling.">Dimethyl fumarate inhibits dendritic cell maturation via nuclear factor kappaB (NF-kappaB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen stress-activated kinase 1 (MSK1) signaling.</a> J Biol Chem. 287, 28017-28026 (2012).</li><li>Li, H. J., Reinhardt, F., Herschman, H. R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-stimulated+mesenchymal+stem+cells+create+a+carcinoma+stem+cell+niche+via+prostaglandin+E2+signaling.">Cancer-stimulated mesenchymal stem cells create a carcinoma stem cell niche via prostaglandin E2 signaling.</a> Cancer Discov. 2, 840-855 (2012).</li><li>Li, X., 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=Intrinsic+resistance+of+tumorigenic+breast+cancer+cells+to+chemotherapy.">Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.</a> J Natl Cancer Inst. 100, 672-679 (2008).</li><li>Diehn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+reactive+oxygen+species+levels+and+radioresistance+in+cancer+stem+cells.">Association of reactive oxygen species levels and radioresistance in cancer stem cells.</a> Nature. 458, 780-783 (2009).</li><li>Hollier, B. G., Evans, K., Mani, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial-to-mesenchymal+transition+and+cancer+stem+cells:+a+coalition+against+cancer+therapies.">The epithelial-to-mesenchymal transition and cancer stem cells: a coalition against cancer therapies.</a> J Mammary Gland Biol Neoplasia. 14, 29-43 (2009).</li><li>Velasco-Velazquez, M. A., Popov, V. M., Lisanti, M. P., Pestell, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+breast+cancer+stem+cells+in+metastasis+and+therapeutic+implications.">The role of breast cancer stem cells in metastasis and therapeutic implications.</a> Am J Pathol. 179, 2-11 (2011).</li><li>Dontu, G., 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=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes Dev. 17, 1253-1270 (2003).</li><li>Charafe-Jauffret, E., 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=Cancer+stem+cells+in+breast:+current+opinion+and+future+challenges.">Cancer stem cells in breast: current opinion and future challenges.</a> Pathobiology. 75, 75-84 (2008).</li><li>Clayton, H., Titley, I., Vivanco, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+progenitor/stem+cells+derived+from+the+human+mammary+gland.">Growth and differentiation of progenitor/stem cells derived from the human mammary gland.</a> Exp Cell Res. 297, 444-460 (2004).</li><li>Ginestier, 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=ALDH1+is+a+marker+of+normal+and+malignant+human+mammary+stem+cells+and+a+predictor+of+poor+clinical+outcome.">ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.</a> Cell Stem Cell. 1, 555-567 (2007).</li><li>Rosen, J. M., Jordan, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increasing+complexity+of+the+cancer+stem+cell+paradigm.">The increasing complexity of the cancer stem cell paradigm.</a> Science. 324, 1670-1673 (2009).</li><li>Visvader, J. E., Lindeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+in+solid+tumours:+accumulating+evidence+and+unresolved+questions.">Cancer stem cells in solid tumours: accumulating evidence and unresolved questions.</a> Nat Rev Cancer. 8, 755-768 (2008).</li></ol>

The authors have nothing to disclose.

In this protocol, we demonstrated the synthesis of an ASA prodrug, GTCpFE, where the fumarate pharmacophore was incorporated to improve the anti-NF&#312;B activity in breast cancer cells. GTCpFE is an effective NF&#312;B inhibitor, whereas ASA itself is not, even at much higher concentrations. The fumarate moiety has anti-inflammatory properties as shown by its ability to inhibit NF&#312;B signaling in a variety of cell lines and tissues14,25-29. The prodrug strategy described herein, is amendable to other malignancies where multiple inflammatory pathways are active and contribute to the pathology. Therefore, GTCpFE may be the prototype for developing new fumarate-based anti-inflammatory and anti-CSC class of drugs. Besides ASA, other non-steroidal anti-inflammatory drugs may also be used. In this case, the synthetic route should be re-designed as well. Ensuring that the new prodrug maintains its anti-NF&#312;B and anti-cyclooxygenase activity via the assays describe herein is critical. 
GTCpFE abrogated MS formation and inhibited the CD44+CD24- immunophenotype. Together these findings suggest that GTCpFE may also be a promising, clinically relevant anti-inflammatory molecule for eradicating breast CSCs by exploiting CSC's reliance on multiple inflammatory pathways, including the NF&#312;B pathway and the cyclooxygenase 2-prostaglandin E2 axis16-21,30. Targeting breast CSCs is important because they are thought to contribute to therapy resistance, recurrence and metastasis31-34. 
The anti-CSCs properties described here, the mammosphere formation and the CD44+CD24- immunophenotype, are based on typical in vitro assays to assess effect on stemness. Mammosphere formation is a functional assay because it exploits the unique property of stem-like/progenitor cells to survive and grow in serum-free suspension, while more differentiated cells undergo anoikis and die in these conditions35,36. However, because a reduction in mammosphere formation may represent (i) increased apoptosis, (ii) decreased proliferation of early progenitor cells or stem cells, (iii) a reduction in stem cell self-renewal, or (iv) interference with anchorage-independent growth, it is best used in conjunction with other anti-CSC assays. The choice of the well-established breast CSC CD44+CD24- immunophenotype is based on the pioneering study by Clarke and colleagues who used breast cancer xenografts to isolate a population of cells capable of initiating tumors in immunodeficient mice24. Similar to the CD44+CD24- immunophenotype, expression of other cell surface markers can be used to isolate stem cells via flow cytometry, but the choice of marker can greatly vary depending on tissues or species (human vs murine model). Alternative assays to measure effects on breast CSCs in vitro are (i) the side population technique, which is based on the ability of stem cells to exclude vital dyes via transmembrane transporters37, and (ii) the ALDEFLUOR assay, which is based on the enzymatic activity of aldehyde dehydrogenase 1 (ALDH1)38. Lastly, a suppression of pluripotency genes, transcription factors, or key stemness pathways may also be indicative of anti-CSCs effects. Promising in vitro anti-CSC properties should be followed up by in vivo characterization. The "gold standard" for assaying anti-CSC properties is in vivo tumorigenicity, wherein the ability of drug-treated cells to initiate or seed a xenograft tumor is examined39,40.

Inflammation is a hallmark that underlies multiple aspects of tumorigenesis, such as incidence and promotion, and eventually progression to metastasis1. In breast cancer, this is further supported by epidemiological observations showing that regular use of the classical non-steroidal anti-inflammatory drug aspirin (acetyl salicylic acid, ASA) is associated with a reduction in both breast cancer incidence, and risk of metastasis and recurrence2,3. ASA acts primarily by inhibiting cyclooxygenase-2 activity, which is often upregulated in breast cancer4,5. However, the anti-cancer effects of ASA may also be mediated by suppressing aberrant nuclear factor &#954;B (NF&#954;B) signaling6-8. This is important because a deregulated NF&#954;B pathway promotes tumor cell survival, proliferation, migration, invasion, angiogenesis, and resistance to therapy9-11. NF&#954;B pathway activation is also critical for mounting an immune response. Therefore, for anti-cancer therapy where prolonged NF&#954;B inhibition is required, one must consider the detrimental side effects involving long-lasting immune suppression. Hence, ASA may serve as a good starting point for therapeutic optimization.
One limitation for ASA application in cancer therapy is the elevated doses required for cyclooxygenase 2 and NF&#954;B inhibition, which are associated with gastrointestinal toxicity, such as ulcers and stomach bleeding12,13. However, converting ASA into as ester prodrug, may reduce ASA&#39;s gastrointestinal toxicity. To further enhance potency and/or add functionality, additional structural elements or ancillary pharmacophores may also be incorporated into ester prodrug design. One such pharmacophore added to enhance ASA potency against the NF&#954;B pathway is fumarate, which we have previously shown to be important for NF&#954;B pathway inhibition14,15.
We synthesized an aspirin-fumarate prodrug15, GTCpFE, and hypothesized that such hybrid molecule would be safe yet potent against the NF&#954;B pathway. We tested its anti-NF&#954;B activity in breast cancer cells and its ability to block breast cancer stem cells (CSCs)15, which rely on NF&#954;B signaling for survival and growth16-21. We find that the potency of GTCpFE against the NF&#954;B pathway is significantly improved over ASA15. In addition, GTCpFE blocks mammosphere formation and attenuates the CSC surface marker CD44+CD24- immunophenotype, indicating that GTCpFE is capable of eradicating CSCs15. These results establish the aspirin-fumarate prodrug as an effective anti-inflammatory agent that can also target breast CSCs. In terms of breast cancer therapy, GTCpFE may have the potential to treat aggressive and deadly disease.

<table><tbody><tr><td>RPMI 1640 Red Medium</td><td>Life Technologies&amp;nbsp;</td><td>11875-093</td><td>Warm up to 37 &amp;deg;C before use</td></tr><tr><td>IMEM</td><td>Corning</td><td>10-024-CV</td><td>Warm up to 37 &amp;deg;C before use</td></tr><tr><td>DMEM / F12 Medium</td><td>ThermoFisher</td><td>21041-025</td><td>Warm up to 37 &amp;deg;C before use</td></tr><tr><td>MEM NEAA 10 mM 100x</td><td>Life Technologies&amp;nbsp;</td><td>11140</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies&amp;nbsp;</td><td>15140-122</td><td></td></tr><tr><td>L-Glutamine 100x</td><td>Life Technologies&amp;nbsp;</td><td>25030-081</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I-1882</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Trypsin 2.5% 10x</td><td>Invitrogen</td><td>15090046</td><td></td></tr><tr><td>Methyl Cellulose</td><td>Sigma&amp;nbsp;</td><td>M0262</td><td></td></tr><tr><td>B27 Supplement 50x</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>EGF</td><td>Gibco</td><td>PHG0311L</td><td></td></tr><tr><td>NF&amp;kappa;B-RE Luciferase Construct&amp;nbsp;</td><td>Clontech&amp;nbsp;</td><td>pGL4.32</td><td></td></tr><tr><td>Renilla Luciferase Construct&amp;nbsp;</td><td>Promega</td><td>pGL4.70</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>ThermoFisher</td><td>11668-019</td><td></td></tr><tr><td>Dual-Luciferase Reporter Assay&amp;nbsp;</td><td>Promega</td><td>120000032</td><td></td></tr><tr><td>NanoDrop Spectrophotometer</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Eppendorf Mastercycler&amp;nbsp;</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>StepOne Real Time PCR System</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Eclipse Microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>CyAn ADP Analyzer&amp;nbsp;</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>QCapture Software</td><td>QImaging</td><td></td><td></td></tr><tr><td>Summit Software</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>GraphPad Software</td><td>Prism</td><td></td><td></td></tr><tr><td>TRIzol</td><td>ThermoFisher</td><td>15596-018</td><td></td></tr><tr><td>M-MLV Reverse Transcriptase</td><td>Invitrogen</td><td>28025-013</td><td></td></tr><tr><td>100 mM dNTP Set</td><td>Invitrogen</td><td>10297-018</td><td></td></tr><tr><td>Random Hexamers&amp;nbsp;</td><td>Invitrogen</td><td>48190-011</td><td></td></tr><tr><td>Fast SYBR Green Master Mix</td><td>ThermoFisher</td><td>4385612</td><td></td></tr><tr><td>Costar 96W, ultra low attachment&amp;nbsp;</td><td>Corning</td><td>3474</td><td></td></tr><tr><td>HBSS, 1x</td><td>ThermoFisher</td><td>14025134</td><td></td></tr><tr><td>CD44-APC conjugated antibody&amp;nbsp;</td><td>BD Pharmingen</td><td>560990</td><td>Store at 4 &amp;deg;C, protect from light</td></tr><tr><td>CD24-PE antibody</td><td>BD Pharmingen</td><td>560991</td><td>Store at 4 &amp;deg;C, protect from light</td></tr><tr><td>Recombinant Human TNF&amp;alpha;</td><td>Fisher</td><td>210TA100&amp;nbsp;</td><td></td></tr><tr><td>CCL2 Primer Forward Sequence</td><td></td><td></td><td>AGAATCACCAGCAGCAAGTGTCC</td></tr><tr><td>CCL2 Primer Reverse Sequence</td><td></td><td></td><td>TCCTGAACCCACTTCTGCTTGG</td></tr><tr><td>ICAM1 Primer Reverse Sequence</td><td></td><td></td><td>TGACGAAGCCAGAGGTCTCAG</td></tr><tr><td>ICAM1 Primer Forward Sequence</td><td></td><td></td><td>AGCGTCACCTTGGCTCTAGG</td></tr><tr><td>TNF Primer Forward Sequence</td><td></td><td></td><td>AAGGGTGACCGACTCAGCG</td></tr><tr><td>TNF Primer Reverse Sequence</td><td></td><td></td><td>ATCCCAAAGTAGACCTGCCCA</td></tr><tr><td>36B4 Primer Forward Sequence</td><td></td><td></td><td>GTGTTCGACAATGGCAGCAT</td></tr><tr><td>36B4 Primer Reverse Sequence</td><td></td><td></td><td>GACACCCTCCAGGAAGCGA</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S5881-500G</td><td></td></tr><tr><td>4-Hydroxybenzyl Alcohol</td><td>Sigma-Aldrich</td><td>20806-10G</td><td></td></tr><tr><td>&lt;em&gt;O&lt;/em&gt;-Acetylsalicyloyl Chloride</td><td>Sigma-Aldrich</td><td>165190-5G</td><td></td></tr><tr><td>Phosphorous Pentoxide</td><td>Sigma-Aldrich</td><td>79610-100G</td><td></td></tr><tr><td>Ethyl Fumaroyl Chloride</td><td>Sigma-Aldrich</td><td>669695-1G</td><td></td></tr><tr><td>Sodium Sulfate</td><td>Sigma-Aldrich</td><td>246980-500G</td><td></td></tr><tr><td>4-Dimethylaminopyridine</td><td>Sigma-Aldrich</td><td>714844-100ML</td><td>0.5&amp;nbsp;M in tetrahydrofuran</td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich</td><td>T0886-100ML</td><td></td></tr><tr><td>Toluene</td><td>Sigma-Aldrich</td><td>244511-100ML</td><td></td></tr><tr><td>Ethyl Acetate</td><td>Sigma-Aldrich</td><td>270989-100ML</td><td></td></tr><tr><td>Tetrahydrofuran</td><td>Sigma-Aldrich</td><td>401757-2L</td><td></td></tr><tr><td>400 MHz FT NMR spectrometer&amp;nbsp;</td><td></td><td></td><td>See Bruker&amp;rsquo;s Avance User&amp;rsquo;s Manual, version 000822 for details</td></tr></tbody></table>

synthesis and characterization of an aspirin-fumarate prodrug that inhibits nf&#954;b activity and breast cancer stem cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

In Figure 1, the chemical structure of aspirin-fumarate prodrug, GTCpFE, and its inhibitory activity on cytokine induced NF&#312;B pathway in breast cancer cells are indicated. GTCpFE inhibits both NF&#312;B endpoints, NF&#312;B-RE luciferase activity (Figure 1B) and expression of NF&#312;B target genes, such as Intercellular Adhesion Molecule 1 (ICAM1), Chemokine C-C Motif Ligand 2 (CCL2), and Tumor Necrosis Factor (TNF) (Figure 1C) in MCF-7 breast cancer cells. Calculated inhibitory concentration at 50% (IC50) value on both endpoints is ~20 &#181;M. IC50 value is calculated using a graphing software. By comparison ASA itself even at 200 &#181;M (10x IC50 of GTCpFE) shows no inhibitory activity (Figure 1B, blue line) in breast cancer cells. This indicates that the prodrug strategy of adding fumarate pharmacophore to ASA significantly improves its anti-NF&#312;B activity.
To measure the anti-CSC activity, we used two assays: the mammosphere (MS) formation assay and the population of cells expressing the CD44+CD24- immunophenotype, a bona fide CSC surface marker in breast cancer24. GTCpFE inhibits MS formation of MDA-MB-231 breast cancer cells in a dose dependent manner shown in Figure 2A. Similar to NF&#312;B pathway inhibition in adherent cultures, the IC50 value for mammosphere formation is ~20 &#181;M. This consistency is expected, given that CSCs rely on NF&#312;B signaling for survival and propagation16-21. In addition, GTCpFE pre-treatment resulted in a significant depletion of the CD44+CD24- population (Figure 2B) in MDA-MB-231 cells. Together, these results establish GTCpFE&#39;s ability to effectively target breast CSCs.
<img alt="Figure 1" src="/files/ftp_upload/54798/54798fig1.jpg" /> 
Figure 1: GTCpFE inhibits the NF&#312;B pathway in breast cancer cells. (A) The chemical structure of the aspirin-fumarate prodrug, GTCpFE. (B-C) MCF-7 cells were pre-treated for 2 hr with various concentrations of GTCpFE, ASA, or vehicle followed by treatment with TNF&#945; (10 ng/ml) for 2-4 hr. (B) NF&#954;B-RE activity was measured by dual luciferase reporter assay. (C) Expression of NF&#954;B target genes, ICAM1, CCL2 and TNF was measured by RT-QPCR. Drug inhibitory activity is plotted as % of TNF&#945; alone. Data are presented as mean &#177; SEM. This figure has been modified from reference15. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54798/54798fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54798/54798fig2.jpg" /> 
Figure 2: GTCpFE inhibits the mammosphere formation and the CD44+CD24- immunophenotype in breast cancer cells. (A) Mammosphere (MS) formation of MDA-MB-231 cells was measured after treatment with varying concentrations of GTCpFE. Quantitation of MS growth (left) and representative pictures of MS at 20X (right) are shown. The effect of GTCpFE is plotted as % vehicle control. (B) The CD44+CD24- population was determined by FACS analysis of MDA-MB-231 cells treated with 50 &#181;M GTCpFE for 72 hr. Quantitation of each population percentage (left) and representative scatter plots from FACS (right) are shown. Data are presented as mean &#177; SEM and statistical analysis of 2-way ANOVA followed by Tukey posttest. *** P&#60;0.001. This figure has been modified from reference15.

Watch this Scientific Journal Video about Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFÎºB Activity and Breast Cancer Stem Cells at JoVE.com

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

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12.2K Views.  University of Illinois at Chicago. This procedure will demonstrate how we synthesized and characterized the anti-NFκB and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

Video: Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFκB Activity and Breast Cancer Stem Cells

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

Chemistry

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.

We have synthesized a novel analogue of pancratistatin with comparable anti-cancer activity as native pancratistatin; interestingly, combinatory treatment with tamoxifen yielded a drastic enhancement in apoptotic and autophagic induction by mitochondrial targeting with minimal effect on noncancerous fibroblasts. Thus, JCTH-4 in combination with tamoxifen could provide a safe anti-cancer therapy.

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce ...

Medicine

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Biology

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFκB Activity and Breast Cancer Stem Cells

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