Name: screening bioactive nanoparticles in phagocytic immune cells for inhibitors of toll-like receptor signaling
Uploaded: 2017-07-26T14:00:00
Description: Watch this Scientific Journal Video about Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling at JoVE.com

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
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	<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.
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		<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, ad...

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	<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>

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 c...

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 na...

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

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 ...

The overall goal of this screening procedure is to identify novel bioactive nanoparticles that can inhibit toll-like receptor signaling. This method can help you physically identify novel nanoinhibitors targeting toll-like receptor signaling in innate immune cells. The main advantage of this technique is that it is fast and robust and easy to perform using reporter cell systems.The applications of this technique extend toward the therapy of many inflammatory diseases because toll-like receptor signaling is found to be involved in the pathogenesis of these diseases. Though this method can provided high screening on and gold nanoparticles, it can also be applied to other nanoparticle systems as well as therapeutic small molecules and the biologies. To begin this procedure, prepare the culture mediums as outlined in the text protocol.Next, add 100 milliliters of ultra-pure endotoxin-free water to a clean 125 milliliter glass flask. Add one pouch of secreted embryonic alkaline phosphatase substrate powder, and swirl the solution gently until it is fully dissolved. Incubate at 37 degrees Celsius for one hour to ensure complete dissolution of the substrates.Using a 0.2 micrometer membrane, filter the solution. Store at four degrees Celsius until ready to use. Next, add one pouch of luciferase substrate powder to a 50 milliliter centrifuge tube containing 25 milliliters of ultra pure endotoxin-free water.After the powder has dissolved completely, store at four degrees Celsius for up to a week until ready to use. Then, prepare the stock solutions of all other reagents as outlined in the text protocol. After culturing and preparing the cells, transfer them from the culture flask to a centrifuge tube.Spin down the cells at 300 times G for five minutes. Then, resuspend them in the R-10 medium at a concentration of 1 X 10 to the 6 cells per milliliter. Add aliquots of the PMA solution to the cell suspension such that the final concentration is 50 nanograms per milliliter.Pour the cell suspension into a sterile reagent reservoir. Using a multi-channel pipette, transfer 100 microliters of the cell suspension into each well of a 96-well flat bottom culture plate. Incubate in a cell culture incubator for 24 hours at 37 degrees Celsius.After this, use a vacuum aspirator to carefully remove the culture medium. Using an inverted microscope, check the cells to ensure that the derived macrophages are adhered at the bottom of the well. Gently wash the cells twice using 100 microliters of PBS per well for each wash.Then, add 100 microliters of fresh R-10 medium to each well. Let the cells rest for two days in an incubator at 37 degress Celsius before conducting the reporter assay. First, determine the optimal LPS dose as outlined in the text protocol.Next, add 180 microliters of pre-warmed Quanti-Blue solution to each well of a new 96-well flat bottom plate. Then, transfer 20 microliters of supernatant from each sample into the plate. Incubate at 37 degrees Celsius for one to two hours to allow for the color to develop from pink to dark blue.After the optical density is above one, use a plate reader to determine the absorption at 655 nanometers. To begin analysis of IRF activation, transfer 10 microliters of fresh supernatant from each sample into a fresh 96-well clear flat bottom white plate. Using auto-injection, add 50 microliters of the luciferase solution to each well.Then immediately collect the luminescence well by well to produce a dose response curve to determine the optimal LPS concentration for nanoparticle screening. Next, centrifuge 20 volumes of the peptide-GMP hybrid solution in 1.5 milliliter Eppendorf tubes at 18, 000 times G for 30 minutes. Carefully discard the supernatants.Transfer the hybrids to a single fresh tube and wash them twice with one milliliter of PBS. After this, resuspend the washed hybrids in one volume of R-10 medium. Mix equal volumes of the concentrated hybrids and the LPS containing R-10 medium such that the final concentration of the hybrids and LPS are 100 nanomolar and 10 nanograms per milliliter, respectively.Next, remove the culture medium from the macrophage culture plate. Add 100 microliters of the hybrid LPS mixture into each well, with three replicates for each condition. Include a negative control and an LPS control.Incubate at 37 degrees Celsius for 24 hours. Then, transfer the medium of each well to a separate centrifuge tube. Centrifuge the tubes at 18, 000 times G and four degrees Celsius for 30 minutes.Transfer the supernatants into a fresh 96-well round bottom plate. To begin the reporter assay on these samples, add 180 microliters of pre-warmed Quanti-Blue solution to each well of a new 96-well flat bottom plate. Then, transfer 20 microliters of the supernatants from each sample into this plate.Incubate at 37 degrees Celsius for one to two hours until the dark blue color develops to an optical density over one. After this, use a plate reader to record the absorption at 655 nanometers. Next, transfer 10 microliters of fresh supernatant from each sample into a 96-well clear flat bottom white plate.Using an automatic injector, add 50 microliters of the luciferase solution to each well, and immediately record the luminescence well by well. Then, validate the inhibitory effect of the potential candidates, and evaluate the TLR specificity as outlined in the text protocol. In this study, the activation of NF-Kappa B AP1 is detected by the SEAP colorimetric assay, in which TLR-4 activation by LPS results in the activation of NF-Kappa B AP1 and the production of SEAP.The released SEAP converts the substrate, leading to a color change that is proportional to the amount of SEAP released upon stimulation. This is quantified by measuring the absorbance at 655 nanometers. Next, LPS-mediated activation of IRF led to the expression of luciferase, catalyzing the substrate to produce luminescence.An optimal LPS concentration of 10 nanograms per milliliter is determined from these dose responses to screen a previously established library of peptide-GMP hybrids. From this, a group of hybrids is identified for their potent ability to inhibit LPS-triggered activation for both NF-Kappa B AP1 and IRF. Validation of this inhibitory activity shows that as the concentration of LPS increases, the inhibitory effects of the hybrid reduce, as expected.Immunoblotting is then conducted to confirm inhibition activity. The lead hybrid is seen to reduce p65 phosphorylation, inhibit I-Kappa B alpha degradation, and delay IRF3 phosphorylation, confirming that the lead hybrid, P12, is able to inhibit LPS-mediating TLR-4 signaling. Additional investigation reveals that P12 also reduces TLR-2 and TLR-5-mediated NF-Kappa B AP1 signaling, as well as TLR-3-mediated IRF activation, suggesting that P12 has potent inhibitory activity on multiple TLR pathways.Following this procedure, other measures like immunoblotting and the cytotoxicity assay is to be performed in order to validate the screening results and to avoid false positive discovery. After its development, this technique paved the way for other researchers in the field of nanomedicine to explore nanoinhibitors in toll-like receptor signaling as next-generation and informatory therapeutics.

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SCIENTISTS IN ACTION

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  ...

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.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...