The authors would like to acknowledge the support from the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (H.Y.), the starting fund from Shanghai First People&#39;s Hospital (H.Y.), Gaofeng Clinical Medicine Grant support from Shanghai Jiaotong University School of Medicine (H.Y.), and the funding from the Crohn&#39;s and Colitis Foundation of Canada (CCFC) (S.E.T. and H.Y.).

1. Preparation of Cell Culture Media and Reagents
<ol>
	<li>Prepare the complete cell culture medium R10 by adding the supplements of 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM sodium pyruvate into the RPMI-1640 medium.
	<ol>
		<li>Prepare the selection culture medium R10-Z by adding the antibiotics Zeocin (200 &#956;g/mL) to R10 for maintaining the expression of SEAP under the control of NF-&#954;B/AP-1 activation. To select for cells expressing both SEAP and luciferase reporter genes, add both Zeocin (100 &#956;g/mL) and blasticidin (10 &#956;g/mL) to R10 (as R10-ZB).</li>
	</ol>
	</li>
	<li>Prepare the SEAP substrate solution by dissolving one pouch of the substrate powder (e.g., QUANTI-Blue) into 100 mL ultrapure, endotoxin free water in a clean 125 mL glass flask.
	<ol>
		<li>Swirl the solution gently and incubate it at 37 &#176;C for 1 h to ensure the complete dissolution of the substrates.</li>
		<li>Filter the solution using a 0.2 &#956;m membrane to ensure its sterility (optional), and store it at 4 &#176;C up to 2 weeks prior to use.</li>
	</ol>
	</li>
	<li>Prepare the luciferase substrate solution by dissolving one pouch of the substrate powder (e.g., QUANTI-Luc) into 25 mL ultrapure, endotoxin free water in a sterile 50 mL centrifuge tube.
	<ol>
		<li>After dissolving the powder completely, use the solution immediately. Alternatively, store the solution at 4 &#176;C (up to a week) or at -20 &#176;C (up to a month) prior to use. 
		CAUTION: Both substrate solutions are light sensitive, and should avoid light exposure whenever is possible. Multiple freeze-thaw cycles of the solution can cause instability of the substrate and shorten its shelf-life.</li>
	</ol>
	</li>
	<li>Prepare the stock solution of phorbol 12-myristate 13-acetate (PMA) in molecular grade dimethyl sulfoxide (DMSO) to have a concentration of 500 &#956;g/mL. Make aliquots of the stock solution (10 &#956;L in a 500 &#956;L tube) and store them at -20 &#176;C.</li>
	<li>Prepare LPS (E-coli K12) stock solution in sterile, endotoxin free water at a concentration of 5 mg/mL, and make aliquots for long-term storage at -20 &#176;C. Prepare a working LPS solution by diluting the stock LPS into phosphate buffered saline (PBS) at a concentration of 100 &#956;g/mL and store at -20 &#176;C prior to use. 
	CAUTION: The reagent preparation for culture uses should be carried out in a biosafety cabinet, and all the containers should be sterilized prior to use. Repeated freeze-thaw cycles of PMA and LPS solutions should be avoided.</li>
</ol>
2. Culture of THP-1 reporter cell-derived macrophages
<ol>
	<li>Use two THP-1 reporter cell lines: THP-1-XBlue and THP-1-Dual cells. The former has a SEAP reporter gene controlled by NF-&#954;B/AP-1, and the latter has a dual reporter gene system, the same SEAP reporter gene and a IRF controlled luciferase gene. Their culture procedures are identical except the selection culture media.
	<ol>
		<li>Thaw a working cell stock (~5 x 106 cells) in 10 mL of R10 medium, spin down the cells at 300 x g for 5 min. Resuspend cells in 10 mL of R10 medium, and transfer them into a T75 culture flask. Exchange the culture medium every 2-3 days until cells reach a density of 1 x 106 cells/mL.</li>
		<li>When cell growth reaches its capacity, passage cells to reduce the cell density to in the range of 2-5 x 105 cells/mL, so the cells can continue to grow.</li>
		<li>After at least 1 passage, start to culture the cells in the selection culture medium: R10-Z for THP-1-XBlue, and R10-ZB for THP-1-Dual. After culturing the cells with selection culture medium for at least 1 passage, cells are ready for the experiment. 
		CAUTION: Cell overgrowing can lead to significant cell death; the cell viability should maintain &#62;98%. Keep a record of the passage number as the cells may behave differently after many passages (&#62; 20 passages). 
		NOTE: Cell culture flasks can be reused several times to save costs; however, this practice may increase the risk of general contamination and cross-contamination among different flasks (if handling different cell lines at the same time). During media exchange and cell passage, the centrifugation is set at 300 x g for 5 min.</li>
	</ol>
	</li>
	<li>To perform reporter cell assay, seed cells into a 96-well flat-bottom cell culture plate and differentiate them into macrophages. The procedures are described in the following.
	<ol>
		<li>Transfer the cells from the flask to a centrifuge tube, spin down the cells at 300 x g for 5 min, and resuspend them in the R10 medium at a concentration of 1 x 106 cells/mL. Add aliquots of the PMA solution into the cell suspensions with a final concentration of 50 ng/mL.</li>
		<li>Transfer 100 &#956;L of the cell suspensions into each well of the 96-well plate using a multi-channel pipette. Incubate the plate at 37 &#176;C (in a cell culture incubator) for 24 h.</li>
		<li>After incubation, carefully remove the culture medium using a vacuum aspirator (or with a multi-channel pipette), and gently wash the cells with PBS (100 &#956;L/well) twice; add 100 &#956;L of fresh R10 medium in each well. Rest the cells for 2 days in an incubator before conducting the reporter assay. 
		CAUTION: The resting step is very important to allow cells to calm down after PMA stimulation to their normal quiet status. This can significantly reduce the background signals of the reporter genes under unstimulated conditions. 
		NOTE: After 24 stimulation with PMA, cells are differentiated into macrophage-like phenotype, with a characteristic of cell adhesion at the bottom of the well, and a morphological feature of pseudopodia.</li>
	</ol>
	</li>
</ol>
3. Screening for Potential TLR4 Nano-inhibitors Using the Reporter Cells
NOTE: Since TLR4 signaling utilizes both MyD88-dependent and TRIF-dependent pathways, it is selected as the primary target to encompass a wide range of TLR signaling pathways. THP-1-XBlue reporter cells are used to mainly examine the NF-&#954;B/AP-1 activation while THP-1-Dual cells are for IRF activation from the TRIF-dependent signal transduction.
<ol>
	<li>Identify the optimal LPS dose by generating a dose-response curve prior to the screening.
	<ol>
		<li>Dilute the working LPS solution to a final concentration of 0.01 - 100 ng/mL in R10 medium (make dilution in log10 scale). Layout the plate design and make dilution in a 96-well round-bottom culture plate. Make sure that each well has a minimum of 110 &#956;L of solution after dilution.</li>
		<li>Gently remove the culture medium from the cell-seeded plate (from 2.2.3) using a vacuum aspirator.</li>
		<li>Transfer the LPS containing R10 medium prepared in the dilution plate (3.1.1) into the culture plate according to the layout of the samples. Incubate the plate at 37 &#176;C (in a cell culture incubator) for 24 h.</li>
		<li>At 24 h, carefully transfer the supernatants (80 &#956;L/well) into a new 96-well round-bottom plate. Conduct the colorimetric and/or luciferase luminescence assay on these solutions immediately or store them at 4 &#176;C (several hours) or -20 &#176;C (days) prior to the assay development. 
		NOTE: The design of plate layout should be simple and clear for experiment and data analysis. For each condition, 2-4 replicates should be considered. Always include a negative control group (LPS null). It is recommended to use THP-1-XBlue cells for NF-&#954;B/AP-1 activation as the Dual cells have a relatively high background of NF-&#954;B/AP-1 activation after being differentiated into macrophages. However, it is feasible to use the Dual cells for reporting both NF-&#954;B/AP-1 and IRF activation.</li>
	</ol>
	</li>
	<li>Develop the colorimetric assay for the NF-&#954;B/AP-1 activation and the luciferase luminescence assay for IRF activation.
	<ol>
		<li>To assess the NF-&#954;B/AP-1 activation, transfer 20 &#956;L of supernatant of each sample into a new 96-well flat-bottom culture plate; and add 180 &#956;L of pre-warmed SEAP substrate solution into each well. Incubate the plate at 37 &#176;C for 1 - 2 h to allow the color development (pink to dark blue). Collect the absorption at 655 nm on a plate reader. 
		CAUTION: The incubation time can be varied based on the color development (30 min up to overnight incubation). It is recommended to wait until the optical density (O.D.) of the darkest color reaches above 1, while avoiding the saturation of the color development (O.D &#62;3).</li>
		<li>For the analysis of IRF activation, transfer 10 &#956;L of supernatant of each sample into a 96-well clear flat-bottom white plate. Add the luciferase solution (50 &#956;L) and immediately collect the luminescence well by well. 
		NOTE: It is highly recommended to use a luminescence plate reader with an auto-injection function to ensure the consistency in the luminescence reading from well to well and from plate to plate. If the substrate solutions are manually injected, please ensure a consistent &#160;incubation time and reading set-up for each well.</li>
	</ol>
	</li>
	<li>Conduct the screening assay on various peptide-GNP hybrids with 10 ng/mL LPS stimulation (based on 3.1). Follow the same procedure in 3.2 for the reporter assay development.
	<ol>
		<li>Concentrate the peptide-GNP hybrids to 200 nM in R10 medium using the centrifugation method. Centrifuge 20 volumes of the hybrid solution (10 nM) at 18,000 x g for 30 min, and carefully discard the supernatants. Collect the hybrids (at the bottom of the tube) into a tube, wash them with PBS twice, and re-suspend the hybrids in one volume of the R10 medium.</li>
		<li>Mix equal volumes of the concentrated hybrids and the LPS (20 ng/mL) containing R10 medium to have a final concentration of the hybrids and LPS to be 100 nM and 10 ng/mL, respectively.</li>
		<li>Remove the culture medium from the cultured plate (2.2.3) and add 100 &#956;L of the mixed solution into each well (3 replicates for each condition); include a negative control (medium only) and a LPS control (10 ng/mL LPS without hybrids).</li>
		<li>After 24 h incubation at 37 &#176;C, transfer the medium of each well into a tube and centrifuge the tubes at 18,000 x g, 4 &#176;C for 30 min. Collect the supernatants (50-80 &#956;L/tube) into a 96-well round-bottom plate, and perform the reporter assay as described in 3.2. 
		CAUTION: When discarding the supernatants after centrifugation, do not agitate the hybrids at the bottom of the tube. 
		NOTE: The centrifugation in the step 3.3.4 is very important to remove the non-internalized nanoparticle hybrids from the culture medium, and can avoid the interference of the hybrids with the colorimetric/luminescence readings. This is because the gold nanoparticles can absorb wide wavelengths of light depending on their size and agglomeration.</li>
	</ol>
	</li>
</ol>
4. Validating the Inhibitory Effect of the Potential Candidates
NOTE: To confirm the inhibitory effect of the potential candidates from the screening, two approaches are employed. One is to examine the dose responses of the stimulants (LPS) at a fixed hybrid concentration (or the other way around); the other is to directly look at the inhibition on the NF-&#954;B/AP-1 and IRF3 signals via immunoblotting.
<ol>
	<li>In approach one, perform the reporter assay with 100 nM of the lead hybrids and two LPS concentrations at 1 ng/mL and 10 ng/mL following the same procedures described in 3.3. Include an inactive hybrid (based on the screening results) as a hybrid control for comparison.</li>
	<li>For the immunoblotting approach, please follow the standard experimental procedures.
	<ol>
		<li>Culture THP-1 cells in R10 medium, seed the cells into a 12-well culture plate (2 x 106 cells/well), and differentiate them into macrophages by treating the cells with 50 ng/mL PMA for 24 h followed by resting for 2 days.</li>
		<li>After cell differentiation, stimulate cells with 10 ng/mL LPS with/without the hybrids (100 nM) over time (up to 4 h). At various time points (0, 5, 15, 30, 60, 120 and 240 min), prepare the cell lysates for immunoblotting. Include an inactive hybrid as a control.</li>
		<li>Probe the signals of I&#954;B&#945;, phosphorylated p65, and phosphorylated IRF3 to examine the inhibitory effect of the lead hybrids on the activation of NF-&#954;B and IRF3. Probe the &#946;-actin and total IRF3 signals as internal controls. 
		NOTE: The signal transduction often occurs much faster (in a few hours) then the expression of the reporter enzymes SEAP and luciferase (24 h). It is highly recommended to also perform a viability assay of the tested hybrids at 24 h as another validation method.</li>
	</ol>
	</li>
</ol>
5. Evaluating the TLR Specificity
NOTE: To investigate the TLR specificity of the lead peptide-GNP hybrid, other TLR signaling pathways are tested, including TLR2, TLR3 and TLR5. TLR7, 8 and 9 are excluded because the THP-1 derived macrophages do not respond well to the stimulation of these TLRs due to the lack of TLR7, 8 and 9 expression in macrophages34.
<ol>
	<li>Test various concentrations (1 ng/mL to 25&#160;&#956;g/mL) of the ligands specific to TLR2 (Pam3CSK4), TLR3 (poly I:C) and TLR5 (flagellin) on both reporter cells derived macrophages to obtain the optimal concentration following the same procedure in 3.1 and 3.2.</li>
	<li>Treat the cells with the mixtures of the lead hybrid (100 nM) and each TLR ligand at the concentration obtained from 5.1 to evaluate the inhibitory specificity of the lead hybrid according to the experimental procedure described in 3.3. Include the inactive hybrid as a control for comparison.</li>
</ol>

<ol>
	<li>Akira, S., Takeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signalling.">Toll-like receptor signalling.</a> Nat Rev Immunol. 4 (7), 499-511 (2004).</li><li>Beutler, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLRs+and+innate+immunity.">TLRs and innate immunity.</a> Blood. 113 (7), 1399-1407 (2009).</li><li>Gay, N. J., Symmons, M. F., Gangloff, M., Bryant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+localization+of+Toll-like+receptor+signalling+complexes.">Assembly and localization of Toll-like receptor signalling complexes.</a> Nat Rev Immunol. 14 (8), 546-558 (2014).</li><li>Palm, N. W., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+receptors+and+control+of+adaptive+immunity.">Pattern recognition receptors and control of adaptive immunity.</a> Immunol Rev. 227 (1), 221-233 (2009).</li><li>Kawasaki, T., Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signaling+pathways.">Toll-like receptor signaling pathways.</a> Front Immunol. 5, 461 (2014).</li><li>Uematsu, S., Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+Type+I+interferons.">Toll-like receptors and Type I interferons.</a> J Biol Chem. 282 (21), 15319-15323 (2007).</li><li>Maglione, P. J., Simchoni, N., Cunningham-Rundles, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signaling+in+primary+immune+deficiencies.">Toll-like receptor signaling in primary immune deficiencies.</a> Ann N Y Acad Sci. 1356, 1-21 (2015).</li><li>Drexler, S. K., Foxwell, B. M. <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+toll-like+receptors+in+chronic+inflammation.">The role of toll-like receptors in chronic inflammation.</a> Int J Biochem Cell Biol. 42 (4), 506-518 (2010).</li><li>O'Neill, L. A., Bryant, C. E., Doyle, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+Toll-like+receptors+for+infectious+and+inflammatory+diseases+and+cancer.">Therapeutic targeting of Toll-like receptors for infectious and inflammatory diseases and cancer.</a> Pharmacol Rev. 61 (2), 177-197 (2009).</li><li>Harter, L., Mica, L., Stocker, R., Trentz, O., Keel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+toll-like+receptor-2+and+-4+on+leukocytes+from+patients+with+sepsis.">Increased expression of toll-like receptor-2 and -4 on leukocytes from patients with sepsis.</a> Shock. 22 (5), 403-409 (2004).</li><li>Roger, T., 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=Protection+from+lethal+gram-negative+bacterial+sepsis+by+targeting+Toll-like+receptor+4.">Protection from lethal gram-negative bacterial sepsis by targeting Toll-like receptor 4.</a> Proc Natl Acad Sci U S A. 106 (7), 2348-2352 (2009).</li><li>Tsujimoto, 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=Role+of+Toll-like+receptors+in+the+development+of+sepsis.">Role of Toll-like receptors in the development of sepsis.</a> Shock. 29 (3), 315-321 (2008).</li><li>Blohmke, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR5+as+an+anti-inflammatory+target+and+modifier+gene+in+cystic+fibrosis.">TLR5 as an anti-inflammatory target and modifier gene in cystic fibrosis.</a> J Immunol. 185 (12), 7731-7738 (2010).</li><li>Hartl, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+in+cystic+fibrosis+lung+disease.">Innate immunity in cystic fibrosis lung disease.</a> J Cyst Fibros. 11 (5), 363-382 (2012).</li><li>Celhar, T., Fairhurst, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+in+systemic+lupus+erythematosus:+potential+for+personalized+treatment.">Toll-like receptors in systemic lupus erythematosus: potential for personalized treatment.</a> Front Pharmacol. 5, 265 (2014).</li><li>Chen, J. Q., Szodoray, P., Zeher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-Like+Receptor+Pathways+in+Autoimmune+Diseases.">Toll-Like Receptor Pathways in Autoimmune Diseases.</a> Clin Rev Allergy Immunol. 50 (1), 1-17 (2016).</li><li>Hennessy, E. J., Parker, A. E., O'Neill, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Toll-like+receptors:+emerging+therapeutics.">Targeting Toll-like receptors: emerging therapeutics.</a> Nat Rev Drug Discov. 9 (4), 293-307 (2010).</li><li>Hedayat, M., Netea, M. G., Rezaei, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+Toll-like+receptors:+a+decade+of+progress+in+combating+infectious+diseases.">Targeting of Toll-like receptors: a decade of progress in combating infectious diseases.</a> Lancet Infect Dis. 11 (9), 702-712 (2011).</li><li>Huang, 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=Engineering+DNA+nanoparticles+as+immunomodulatory+reagents+that+activate+regulatory+T+cells.">Engineering DNA nanoparticles as immunomodulatory reagents that activate regulatory T cells.</a> J Immunol. 188 (10), 4913-4920 (2012).</li><li>Lemos, 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=Activation+of+the+STING+adaptor+attenuates+experimental+autoimmune+encephalitis.">Activation of the STING adaptor attenuates experimental autoimmune encephalitis.</a> J Immunol. 192 (12), 5571-5578 (2014).</li><li>Hess, K. L., Andorko, J. I., Tostanoski, L. H., Jewell, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyplexes+assembled+from+self-peptides+and+regulatory+nucleic+acids+blunt+toll-like+receptor+signaling+to+combat+autoimmunity.">Polyplexes assembled from self-peptides and regulatory nucleic acids blunt toll-like receptor signaling to combat autoimmunity.</a> Biomaterials. 118, 51-62 (2017).</li><li>Tostanoski, L. 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=Design+of+Polyelectrolyte+Multilayers+to+Promote+Immunological+Tolerance.">Design of Polyelectrolyte Multilayers to Promote Immunological Tolerance.</a> ACS Nano. , (2016).</li><li>Foit, L., Thaxton, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+high-density+lipoprotein-like+nanoparticles+potently+inhibit+cell+signaling+and+production+of+inflammatory+mediators+induced+by+lipopolysaccharide+binding+Toll-like+receptor+4.">Synthetic high-density lipoprotein-like nanoparticles potently inhibit cell signaling and production of inflammatory mediators induced by lipopolysaccharide binding Toll-like receptor 4.</a> Biomaterials. 100, 67-75 (2016).</li><li>He, C., Hu, Y., Yin, L., Tang, C., Yin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+particle+size+and+surface+charge+on+cellular+uptake+and+biodistribution+of+polymeric+nanoparticles.">Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles.</a> Biomaterials. 31 (13), 3657-3666 (2010).</li><li>Hirn, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+size-dependent+and+surface+charge-dependent+biodistribution+of+gold+nanoparticles+after+intravenous+administration.">Particle size-dependent and surface charge-dependent biodistribution of gold nanoparticles after intravenous administration.</a> Eur J Pharm Biopharm. 77 (3), 407-416 (2011).</li><li>Blanco, E., Shen, H., Ferrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+nanoparticle+design+for+overcoming+biological+barriers+to+drug+delivery.">Principles of nanoparticle design for overcoming biological barriers to drug delivery.</a> Nat Biotechnol. 33 (9), 941-951 (2015).</li><li>Arvizo, R. 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=Modulating+pharmacokinetics,+tumor+uptake+and+biodistribution+by+engineered+nanoparticles.">Modulating pharmacokinetics, tumor uptake and biodistribution by engineered nanoparticles.</a> PLoS One. 6 (9), e24374 (2011).</li><li>Ernsting, M. J., Murakami, M., Roy, A., Li, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+the+pharmacokinetics,+biodistribution+and+intratumoral+penetration+of+nanoparticles.">Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles.</a> J Control Release. 172 (3), 782-794 (2013).</li><li>Lin, P. J., Tam, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+pharmacokinetic/pharmacodynamic+properties+of+therapeutic+nucleotides+using+lipid+nanoparticle+systems.">Enhancing the pharmacokinetic/pharmacodynamic properties of therapeutic nucleotides using lipid nanoparticle systems.</a> Future Med Chem. 7 (13), 1751-1769 (2015).</li><li>Yang, H., Fung, S. Y., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+the+cellular+uptake+of+physiologically+stable+peptide-gold+nanoparticle+hybrids+with+single+amino+acids.">Programming the cellular uptake of physiologically stable peptide-gold nanoparticle hybrids with single amino acids.</a> Angew Chem Int Ed Engl. 50 (41), 9643-9646 (2011).</li><li>Yang, 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=Amino+Acid+Structure+Determines+the+Immune+Responses+Generated+by+Peptide-Gold+Nanoparticle+Hybrids.">Amino Acid Structure Determines the Immune Responses Generated by Peptide-Gold Nanoparticle Hybrids.</a> Particle &amp; Particle Systems Characterization. 30 (12), 1039-1043 (2013).</li><li>Yang, 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=Amino+Acid-Dependent+Attenuation+of+Toll-like+Receptor+Signaling+by+Peptide-Gold+Nanoparticle+Hybrids.">Amino Acid-Dependent Attenuation of Toll-like Receptor Signaling by Peptide-Gold Nanoparticle Hybrids.</a> ACS Nano. 9 (7), 6774-6784 (2015).</li><li>Yang, 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=Endosomal+pH+modulation+by+peptide-gold+nanoparticle+hybrids+enables+potent+anti-inflammatory+activity+in+phagocytic+immune+cells.">Endosomal pH modulation by peptide-gold nanoparticle hybrids enables potent anti-inflammatory activity in phagocytic immune cells.</a> Biomaterials. 111, 90-102 (2016).</li><li>Ospelt, C., Gay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLRs+and+chronic+inflammation.">TLRs and chronic inflammation.</a> Int J Biochem Cell Biol. 42 (4), 495-505 (2010).</li><li>Kuznik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+endosomal+TLR+inhibition+by+antimalarial+drugs+and+imidazoquinolines.">Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines.</a> J Immunol. 186 (8), 4794-4804 (2011).</li><li>Lee, S. J., Silverman, E., Bargman, J. M. <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+antimalarial+agents+in+the+treatment+of+SLE+and+lupus+nephritis.">The role of antimalarial agents in the treatment of SLE and lupus nephritis.</a> Nat Rev Nephrol. 7 (12), 718-729 (2011).</li><li>Mullarkey, 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=Inhibition+of+endotoxin+response+by+e5564,+a+novel+Toll-like+receptor+4-directed+endotoxin+antagonist.">Inhibition of endotoxin response by e5564, a novel Toll-like receptor 4-directed endotoxin antagonist.</a> J Pharmacol Exp Ther. 304 (3), 1093-1102 (2003).</li><li>Barochia, A., Solomon, S., Cui, X., Natanson, C., Eichacker, P. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eritoran+tetrasodium+(E5564)+treatment+for+sepsis:+review+of+preclinical+and+clinical+studies.">Eritoran tetrasodium (E5564) treatment for sepsis: review of preclinical and clinical studies.</a> Expert Opin Drug Metab Toxicol. 7 (4), 479-494 (2011).</li><li>Rossignol, D. 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=Safety,+pharmacokinetics,+pharmacodynamics,+and+plasma+lipoprotein+distribution+of+eritoran+(E5564)+during+continuous+intravenous+infusion+into+healthy+volunteers.">Safety, pharmacokinetics, pharmacodynamics, and plasma lipoprotein distribution of eritoran (E5564) during continuous intravenous infusion into healthy volunteers.</a> Antimicrob Agents Chemother. 48 (9), 3233-3240 (2004).</li><li>Rossignol, D. P., Wong, N., Noveck, R., Lynn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+pharmacodynamic+activity+of+eritoran+tetrasodium,+a+TLR4+antagonist,+during+intermittent+intravenous+infusion+into+normal+volunteers.">Continuous pharmacodynamic activity of eritoran tetrasodium, a TLR4 antagonist, during intermittent intravenous infusion into normal volunteers.</a> Innate Immun. 14 (6), 383-394 (2008).</li><li>Tidswell, 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=Phase+2+trial+of+eritoran+tetrasodium+(E5564),+a+toll-like+receptor+4+antagonist,+in+patients+with+severe+sepsis.">Phase 2 trial of eritoran tetrasodium (E5564), a toll-like receptor 4 antagonist, in patients with severe sepsis.</a> Crit Care Med. 38 (1), 72-83 (2010).</li><li>Opal, S. 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=Effect+of+eritoran,+an+antagonist+of+MD2-TLR4,+on+mortality+in+patients+with+severe+sepsis:+the+ACCESS+randomized+trial.">Effect of eritoran, an antagonist of MD2-TLR4, on mortality in patients with severe sepsis: the ACCESS randomized trial.</a> JAMA. 309 (11), 1154-1162 (2013).</li><li>Fung, S. 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=Unbiased+screening+of+marine+sponge+extracts+for+anti-inflammatory+agents+combined+with+chemical+genomics+identifies+girolline+as+an+inhibitor+of+protein+synthesis.">Unbiased screening of marine sponge extracts for anti-inflammatory agents combined with chemical genomics identifies girolline as an inhibitor of protein synthesis.</a> ACS Chem Biol. 9 (1), 247-257 (2014).</li></ol>

The authors have nothing to disclose.

Since TLRs are involved in the pathogenesis of many inflammatory diseases, they have emerged as therapeutic targets for the modulation of immune responses and inflammatory conditions. However, the clinical development of therapeutics to inhibit TLR signaling pathways has had limited success to date. The antimalarial drug hydroxychloroquine which inhibits TLR7 and TLR9 is in clinical use35,36.&#160;Similarly, only a limited number of compounds have progressed to clinical trials including eritoran, a TLR4 antagonist, that exhibited potent inhibitory effects on LPS-mediated inflammatory responses in pre-clinical studies37,38, showed positive results in the phase I/II clinical trials39,40,41, but ultimately the phase III trial failed to reduce the mortality of patients with severe sepsis42. This failure has many possible reasons, one being that sepsis-associated inflammatory responses are often triggered through multiple TLR pathways, and thus blocking only TLR4 may not be sufficient to reduce the overwhelming inflammation. Therefore, developing novel, potent poly-TLR inhibitors could overcome such clinical challenges and become the next generation anti-inflammatory therapeutics. The screening strategy and protocol described here are expected to serve as an efficient experimental tool in studying TLR signaling, and more importantly as a drug discovery platform to accelerate the search for the next generation TLR inhibitors.
This screening approach provides several advantages in searching for new TLR inhibitors. First, the screening can be achieved in a high-throughput fashion using the reporter cell systems that are fast, sensitive and quantitative. Second, with both reporter cell lines, the screening can be done to cover a wide range of TLR signaling cascades, including the NF-&#954;B/AP-1 pathway and the type I interferon signaling (via IRFs); thus, they are ideal for the screening of multiple TLR pathways. Third, these reporter cells are genetically engineered from the human monocytic THP-1 cell line, which is a good model system to study interventions targeting the innate immune response. Fourth, the cell line is easily maintained, and particularly, can be differentiated into macrophages; and since macrophages play a key role in many disease-associated inflammatory conditions, they serve as an ideal target for the screening of immunotherapeutics targeting TLR signaling. Fifth, monocytes and macrophages have impressive phagocytic capacity, which allows for high cellular uptake of the nanoparticles, making them especially suitable for studying nanoscale therapeutic agents. Furthermore, this screening protocol can be applied to search for not only the nano-based therapeutic agents, but also other types of bio-active compounds.
Although this screening platform is very versatile and robust, some caution must be applied to avoid false discovery. The screening is primarily based on the reporter assay, which relies on the expression of the reporter gene under control of specific signaling events. Ideally, the reporter gene expression (SEAP and luciferase) is proportional to the intensity of the signal transduction pathway of interest, and the impact of the drug candidates is reflected in the assay readouts. However, in reality, any biological events occurring upstream of the reporter gene expression could affect the result, sometimes leading to a false positive discovery. For instance, low expression of SEAP could result from the inhibition of the protein synthesis process rather than from the down regulation of TLR signaling43. To avoid such a false discovery, the inhibitory activity of the identified candidates should always be validated, and the gold standard method is to directly look at the signaling pathways via immunoblotting. Another important aspect in screening nanoparticle-based therapeutic agents is the surface properties of these nanodevices. Based on the surface modifiers, the nanodevices can have various biological activity. However, they may also have non-specific binding capability to certain biomolecules. In the case of non-specific binding to SEAP or luciferase, the catalytic activity to the substrates could be compromised by these nanodevices, leading to potential false discovery. Including extra control groups (e.g., nanodevice only) in the screening reduces the risk of false discovery. Last but not the least, the cytotoxicity of the identified lead candidates must be examined to exclude cytotoxicity as a contributing factor to false discovery. This can be done simultaneously during the screening process using a standard viability assay (e.g., MTS or MTT), or in a separate experiment.

Toll-like receptors (TLRs) are one of the key elements in the innate immune system contributing to the first line of defense against infections. TLRs are responsible for sensing invading pathogens by recognizing a repertoire of pathogen-associated molecular patterns (or PAMPs) and mounting defense reactions through a cascade of signal transduction1,2. There are 10 human TLRs identified; except TLR10 for which the ligand(s) remain unclear, each TLR can recognized a distinct, conserved group of PAMPs. For example, TLR2 and TLR4, primarily located on the cell surface, can detect lipoproteins and glycolipids from Gram-positive and Gram-negative bacteria, respectively; while TLR3, TLR7/8 and TLR9, mainly present in the endosomal compartments, can sense RNA and DNA products from viruses and bacteria3. When stimulated by PAMPs, TLRs trigger essential immune responses by releasing pro-inflammatory mediators, recruiting and activating effector immune cells, and coordinating subsequent adaptive immune events4.
The TLR signaling transduction can be simply categorized into two main pathways5,6. One is dependent upon the adaptor protein myeloid differentiation factor 88 (MyD88) &#8212; the MyD88-dependent pathway. All TLRs except TLR3 utilize this pathway to activate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) and mitogen-associated protein kinases (MAPKs), leading to the expression of pro-inflammatory mediators such as TNF-&#945;, IL-6 and IL-8. The second pathway utilizes TIR-domain-containing adaptor-inducing interferon-&#946; (TRIF) &#8212; the TRIF-dependent or MyD88-independent pathway &#8212; to activate interferon (IFN) regulatory factors (IRFs) and NF-&#954;B, resulting in the production of type I IFNs. Intact TLR signaling is critical to our daily protection from microbial and viral infections; defects in TLR signaling pathways can lead to immunodeficiency and are often detrimental to human health.7
However, TLR signaling is a &#39;double-edged sword&#39; and excessive, uncontrolled TLR activation is harmful. Overactive TLR responses contribute to the pathogenesis in many acute and chronic human inflammatory diseases8,9. For instance, sepsis which is characterized by systemic inflammation and multi-organ injury, is primarily due to acute, overwhelming immune responses toward infections, with TLR2 and TLR4 playing a crucial role in the sepsis pathophysiology10,11,12. In addition, TLR5 has been found to contribute to chronic lung inflammation of patients with cystic fibrosis13,14. Moreover, dysregulated endosomal TLR signaling (e.g., TLR7 and TLR9) is strongly associated with the development and progression of several autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA)15,16. These converging lines of evidence identify TLR signaling as a potential therapeutic target for many inflammatory diseases17.
Although pharmacological regulation of TLR responses is anticipated to be beneficial in many inflammatory conditions, unfortunately, there are currently very few compounds clinically available to inhibit TLR signaling9,17,18. This is partly due to the complexity and redundancy of the TLR pathways involved in the immune homeostasis and disease pathology. Therefore, searching for novel, potent therapeutic agents to target multiple TLR signaling pathways could bridge a fundamental gap, and overcome the challenge of advancing TLR inhibitors into the clinic.
In light of the rapid advances in nanoscience and nanotechnology, nanodevices are emerging as the next generation TLR modulators owing to their unique properties19,20,23. The nanoscale size allows these nano-therapeutics to have better bio-distribution and sustained circulation24,25,26. They can be further functionalized to meet the desired pharmacodynamic and pharmacokinetic profiles27,28,29. More excitingly, the bio-activity of these novel nanodevices arises from their intrinsic properties, which can be tailored for specific medical applications, rather than simply acting as a delivery vehicle for a therapeutic agent. For example, a high-density lipoprotein (HDL)-like nanoparticle was designed to inhibiting TLR4 signaling by scavenging the TLR4 ligand LPS23.&#160;In addition, we have developed a peptide-gold nanoparticle hybrid system, where the decorated peptides can alter the surface properties of the gold nanoparticles, and allow them to have various bio-activities30,31,32,33. This makes them a special class of drug (or &#34;nano-drug&#34;) as the next generation nano-therapeutics.
In this protocol, we present an approach to identify a novel class of peptide-gold nanoparticle (peptide-GNP) hybrids that can potently inhibit multiple TLR signaling pathways in phagocytic immune cells32,33. The approach is based on commercially available THP-1 reporter cell lines. The reporter cells consist of two stable, inducible reporter constructs: one carries a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and activator protein 1 (AP-1); the other contains a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs). Upon TLR stimulation, the signal transduction leads to the activation of NF-&#954;B/AP-1 and/or IRFs, which turns on the reporter genes to secret SEAP and/or luciferase; such events can be easily detected using their corresponding substrate reagents with a spectrophotometer or luminometer. Using this approach to screen our previously established library of peptide-GNP hybrids, we identified lead candidates that can potently inhibit TLR4 signaling pathways. The inhibitory activity of the lead peptide-GNP hybrids was then validated using another biochemical approach of immunoblotting, and evaluated on other TLR pathways. This approach allows for fast, effective screening of novel agents targeting TLR signaling pathways.

<table>
	<tbody>
		<tr>
			<td>THP-1-XBlue reporter cell</td>
			<td>InvivoGen</td>
			<td>thpx-sp</td>
			<td>keep cell culture passage under 20</td>
		</tr>
		<tr>
			<td>THP-1-Dual repoter cell</td>
			<td>InvivoGen</td>
			<td>thpd-nfis</td>
			<td>keep cell culture passage under 20</td>
		</tr>
		<tr>
			<td>RPMI-1640 (no L-glutamine)</td>
			<td>GE Health Care</td>
			<td>SH30096.02</td>
			<td>Warm up to 37 &#176;C before use; add supplements to make a complete medium R10</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (qualified)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12484028</td>
			<td>Heat inactivated; 10% in RPMI-1640</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30034.02</td>
			<td>2 mM in the complete medium R10</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td>1 mM in the complete medium R10</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline, 1X, without calcium, magnesium</td>
			<td>GE Health Care</td>
			<td>SH30028.02</td>
			<td>Use for cell washing and reagent preparation</td>
		</tr>
		<tr>
			<td>QUANTI-Blue</td>
			<td>InvivoGen</td>
			<td>rep-qb1</td>
			<td>SEAP substrate</td>
		</tr>
		<tr>
			<td>QUANTI-Luc</td>
			<td>InvivoGen</td>
			<td>rep-qlc2</td>
			<td>Luciferase substrate</td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>InvivoGen</td>
			<td>ant-zn-1</td>
			<td>Selection antibiotics for reporter cells</td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>InvivoGen</td>
			<td>anti-bl-1</td>
			<td>Selection antibiotics for reporter cells</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO) for molecular biology</td>
			<td>Sigmal-Aldrich</td>
			<td>D8418-100ML</td>
			<td>Use for reagent preparation</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA) for molecular biology</td>
			<td>Sigmal-Aldrich</td>
			<td>P1585-1MG</td>
			<td>Use for cell differentiation</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS) from E. coli K12</td>
			<td>InvivoGen</td>
			<td>tlrl-eklps</td>
			<td>TLR4 ligand</td>
		</tr>
		<tr>
			<td>Pam3CSK4</td>
			<td>InvivoGen</td>
			<td>tlrl-pms</td>
			<td>TLR2/1 ligand</td>
		</tr>
		<tr>
			<td>Poly (I:C) HMW</td>
			<td>InvivoGen</td>
			<td>tlrl-pic</td>
			<td>TLR3 ligand</td>
		</tr>
		<tr>
			<td>Flagellin from S. Typhimurium (FLA-ST), ultrapure</td>
			<td>InvivoGen</td>
			<td>tlrl-epstfla</td>
			<td>TLR5 ligand</td>
		</tr>
		<tr>
			<td>SpectraMax Plus 384 microplate reader</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>Read colorimetric assay</td>
		</tr>
		<tr>
			<td>Infinite M200 Pro multimode microplate reader with injectors</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Read luminiscience</td>
		</tr>
		<tr>
			<td>Microfuge 22R centrifuge</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td>Temperature controlled micro-centrifugator (up to 18,000 g)</td>
		</tr>
		<tr>
			<td>Allegra X-15R centrifuge</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td>Temperature controlled general purpose centrifugator (for cell culture use)</td>
		</tr>
		<tr>
			<td>Costar assay plate, 96-well white with clear flat bottom, tissue culure treated</td>
			<td>Corning Costar</td>
			<td>3903</td>
			<td>Used for luminiscence assay</td>
		</tr>
	</tbody>
</table>

screening bioactive nanoparticles in phagocytic immune cells for inhibitors of toll-like receptor signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

The overall experimental approach is illustrated in Figure 1. The two THP-1 reporter cell lines, THP-1-XBlue and THP-1-Dual, are used to fast screen the TLR responses by probing the activation of NF-&#954;B/AP-1 and IRFs, respectively. The activation of NF-&#954;B/AP-1 can be detected by the SEAP colorimetric assay, whereas IRF activation is monitored by luciferase luminescence. The monocytic THP-1 cells can be easily derived into macrophages to screen the nanodevices for their immunomodulatory activity on the innate phagocytic immune cells. With the reporter system, the screening can be conducted in a high-throughput fashion; such an approach is versatile to the discovery of new immunotherapeutics, particularly targeting on innate immune signaling such as TLR signaling.
The screening procedure and representative results are shown in Figure 2. Briefly, the reporter cells are seeded into a 96-well plate and derived into macrophages (Figure 2A and 2B). Different concentrations of the TLR ligands (TLR4 as an example) are first tested to obtain the optimal concentration for the actual screening of the peptide-GNP hybrids. The TLR4 stimulation by LPS resulted in the activation of NF-&#954;B/AP-1 and the production of SEAP. The released SEAP converted the substrate and changed its photophysical property, which could be monitored by the shifting in the light absorption, leading to the solution color change (Figure 2C). Such a change is proportional to the amount of SEAP released upon stimulation, and can be quantified by measuring the absorbance at 655 nm on a spectrophotometer (Figure 2D). Similarly, the activation of IRFs (triggered by LPS) led to the expression of luciferase, which catalyzed the substrate to produce luminescence (Figure 2E). Based on these dose responses, an optimal concentration of LPS (10 ng/mL) was used to screen a small previously established library of peptide-GNP hybrids (Table 1). The fabrication of the hybrids and their physicochemical characteristics were described in our previous publications30,31,32. From the screening, a group of hybrids (P12 and its derivatives) were identified for their potent inhibitory activity on both NF-&#954;B/AP-1 and IRF activation triggered by LPS (Figure 2F); interestingly, the hybrid P13, just slightly different from P12 in the peptide coatings, did not have any inhibitory activity, which could be served as a hybrid control for comparison. The P13 derivatives showed various degree of mild inhibitory activity depending on the other decorated peptide on the surface.
After identifying the lead hybrid, it is important to validate the inhibitory activity. The inhibition was first confirmed by examining the different ratios of the hybrid to LPS to exclude potential false positive results due to technical artifacts. As the concentration of LPS increased, the inhibitory effect of the hybrid (at a fixed concentration) reduced as expected (Figure 3A and 3B). To further ensure that the observed inhibition from the reporter assays was indeed a result of down-regulating the NF-&#954;B and IRF signaling by the lead hybrid, the immunoblotting was conducted to directly assess the protein signal transduction over time. The activation of NF-&#954;B and IRF3 was examined by probing the phosphorylation of the NF-&#954;B subunit p65 and the degradation of the NF-&#954;B inhibitor I&#954;B&#945;, and phosphorylation of IRF3, respectively. As shown in Figure 3C, the lead hybrid P12 could reduce p65 phosphorylation, inhibit I&#954;B&#945; degradation, and delayed IRF3 phosphorylation, while the inactive hybrid P13 could not (data not shown). All these results confirmed that the identified lead hybrid was able to inhibit LPS-mediated TLR4 signaling by down-regulating both NF-&#954;B and IRF3 activation.
In addition to TLR4 signaling, the inhibitory activity of the lead hybrid was further evaluated on other TLR pathways including TLR2, TLR3 and TLR5 to address the TLR specificity. As shown in Figure 4, the lead hybrid P12 was able to reduce TLR2- and TLR5-mediated NF-&#954;B/AP-1 signaling, as well as TLR3-mediated IRF activation; again, the inactive hybrid P13 did not show any inhibitory activity. These results suggested that the identified lead hybrid has a potent inhibitory activity on multiple TLR pathways.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56075/56075fig1.jpg" /> 
Figure 1: Overall experimental approach of high-throughput screening of TLR inhibitors using the reporter cell assay. Two reporter cell lines are used: THP-1-XBlue and THP-1-Dual. The former has a SEAP reporter gene under the control of NF-&#954;B/AP-1 activation, whereas the Dual system has an additional luciferase reporter gene under the control of IRFs activation. These cells can be easily differentiated into macrophages for high-throughput screening of immune modulatory nanoparticles on innate immune signaling. <a href="https://cloudflare.jove.com/files/ftp_upload/56075/56075fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56075/56075fig2.jpg" /> 
Figure 2: High-throughput screening of nano-inhibitors on TLR4 signaling. 
(A) A scheme of experimental procedures. (B) An optical microscopic image of differentiated macrophages (200x magnification). (C) A representative image of the solution color change from the SEAP reporter assay; the color of the substrate solution turned into purple or dark blue (from original pink) depending on the SEAP expression in the culture medium. (D) Quantitative analysis of the SEAP substrate absorption at 655 nm in response to LPS stimulation. (E) The luciferase luminescence from the IRF reporter system in proportion to LPS stimulation. (F) High-throughput screening identifying the lead peptide-GNP hybrid P12 and its derivatives in inhibiting both NF-&#954;B/AP-1 and IRF pathways of TLR4 signaling; the hybrid concentration = 100 nM; the LPS concentration = 10 ng/mL; the bar represents mean &#177; standard deviation; n = 2. <a href="https://cloudflare.jove.com/files/ftp_upload/56075/56075fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56075/56075fig3.jpg" /> 
Figure 3: Validation of the inhibitory activity of the lead hybrid. Confirmation of the inhibitory effect of the lead hybrid with various concentrations of LPS on both (A) SEAP and (B) luciferase reporter systems. (C) Confirming the inhibition of the lead hybrid on the NF-&#954;B and IRF3 signaling via immunoblotting. The inactive hybrid P13 was used as a hybrid control for comparison. The hybrid concentration = 100 nM; the LPS concentration = 10 ng/mL; the bar represents mean &#177; standard deviation; n = 3; * and *** denote p &#60;0.05 and p &#60;0.001, respectively, using one way ANOVA analysis. <a href="https://cloudflare.jove.com/files/ftp_upload/56075/56075fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56075/56075fig4.jpg" /> 
Figure 4: Inhibitory effect of the lead hybrid on other TLR signaling pathways. 
The inhibition of NF-&#954;B/AP-1 by the lead hybrid following the TLR2 stimulation (Pam3CSK4 = 1 ng/mL) (A) and TL5 stimulation (flagellin = 100 ng/mL) (B). (C) The reduction of both NF-&#954;B/AP-1 and IRF signaling by the lead hybrid following the TLR3 stimulation (poly I:C = 25 &#956;g/mL). The hybrid concentration = 100 nM; the bar represents mean &#177; standard deviation; n = 3; *, **, and&#160;*** denote p &#60;0.05, p &#60;0.01,&#160;and p &#60;0.001, respectively, using one way ANOVA analysis; ns: non-significant. <a href="https://cloudflare.jove.com/files/ftp_upload/56075/56075fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/56075/56075table1.jpg" /> 
Table 1: A small library of peptide-GNP hybrids established in our early studies. 
The hybrids are made of a gold nanoparticle core (~13 nm in diameter) and various hexapeptide coatings on the surface. <a href="https://cloudflare.jove.com/files/ftp_upload/56075/56075table1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling at JoVE.com

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling | Medicine | Video

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

screening-bioactive-nanoparticles-phagocytic-immune-cells-for

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Medicine

Cellular Processes

Cell Signaling

SCIENTISTS IN ACTION

12.3K Views.  Shanghai Jiaotong University School of Medicine. Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling. 

Video: Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.