Name: conditional knockdown of gene expression in cancer cell lines to study the recruitment of monocytes/macrophages to the tumor microenvironment
Uploaded: 2017-11-23T14:00:00
Description: Watch this Scientific Journal Video about Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment at JoVE.com

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

The protocol section that uses human monocytes obtained from healthy volunteers follows the guidelines of the Children&#39;s Hospital Los Angeles Human Research Ethics Committee and has been approved by the Institutional Review Board under the Human Material Protocol number: CCI 08-00208.
1. Preparation of Cancer Cell Lines with Tetracycline-regulated shRNA Expression
<ol>
	<li>Cloning of shRNA PAI-1 into Tet-pLKO-puro vector8,<sup class="x...

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

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

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

<table border="0" cellpadding="0" cellspacing="0" width="868">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tet-pLKO-puro lentiviral vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">21915</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS (Tetracycline-free)</td>
			<td style="width:177px;">Omega Scientific</td>
			<td style="width:119px;">FB-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque</td>
			<td style="width:177px;">Sigma</td>
			<td>10771-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Human Monocyte Enrichment Kit</td>
			<td style="width:177px;">StemCell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Magnet</td>
			<td style="width:177px;">StemCell</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EcoRI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AgeI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3552S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cut Smart buffer</td>
			<td style="width:177px;">NEB</td>
			<td>B7200S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:177px;">Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROMEGA Wizard SV Gel and PCR Cleanup System</td>
			<td style="width:177px;">PROMEGA</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">psPAX vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMD2.G vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12259</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:405px;">One Shot Stbl3 Chemically Competent&#160;E. coli</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C737303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hema 3 stain set for Wright-Giemsa stain</td>
			<td style="width:177px;">Protocol</td>
			<td style="width:119px;">123-869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK293 cells</td>
			<td style="width:177px;">ATCC</td>
			<td style="width:119px;">CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XhoI restriction enzyme</td>
			<td style="width:177px;">NEB</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ligase</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ligase buffer</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">EDTA 0.5 M solution</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Lipofectamine 2000</td>
			<td style="width:177px;">Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">The Big Easy&#34; EasySep Magnet</td>
			<td style="width:177px;">Stem Cell</td>
			<td style="width:119px;">18001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.1% Poly-L-Lysine solution</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Acetate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium butyrate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;M syringe filters</td>
			<td style="width:177px;">VWR International</td>
			<td>28145-479</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:119px;">15504-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride(NaCl)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653-5 kg</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dithioerythritol (DTE)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B8255-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol (EtOH)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">792780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tryptone</td>
			<td style="width:177px;">Amresco</td>
			<td style="width:119px;">J859-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">yeast extract</td>
			<td style="width:177px;">Fluka</td>
			<td style="width:119px;">70161-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:177px;">BD</td>
			<td style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ampicillin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate-buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycycline hyclate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9891-25g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roswell Park Memorial Institute (RPMI)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.5 M EDTA pH 8.0</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Falcon Permeable Support for 24 Well Plate with 8.0&#956;m Transparent PET Membrane</td>
			<td style="width:177px;">Becton Dickinson</td>
			<td style="width:119px;">353097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Bovine Serum Albumin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton-Tipped Applicator&#160;</td>
			<td style="width:177px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol Hema 3 STAT pack&#160;</td>
			<td style="width:177px;">Fisher Scientific Company</td>
			<td style="width:119px;">123-869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Resolve Immersion Oil, High Viscosity</td>
			<td style="width:177px;">Richard Allan Scientific</td>
			<td style="width:119px;">M4004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Penicillin Streptomycin</td>
			<td style="width:177px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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

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

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

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

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

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

The overall of this protocol is to utilize conditional knockdown of gene expression in cancer cells to study the recruitment of monocytes and macrophages to the tumor microenvironment in vitro. This method can help answer key questions in the tumor microenvironment field regarding the nature of the cross stock between the cancer cells and the cells of the immune system. The main advantage of this technique is that the system used here requires single vector cloning, allows quick generation of multiple clones, and exhibits very low levels of thickness.Also demonstrated is a method for purification of human monocytes without direct contact with antibodies which avoids accidental activation while resulting in more than 95%pure population of human monocytes. To produce viral particles, transfect 70%confluent 150 millimeter dishes of HEK-293 cells with 25 micrograms of pLKO-tet-on shRNA, 25 micrograms of the psPAX packaging plasmid, and five micrograms of the envelope-expressing plasmid pMD2. G using transfection reagent as per standard protocols.Add mix to the 150 millimeter dishes. The next day, induce Lentiviral production in the cells by changing the medium to DMEM supplemented with 10 millimolar sodium butyrate and 20 millimolar HEPES. Culture for eight hours at 37 degrees Celsius and 5%carbon dioxide.After eight hours, wash the cells with PBS and add 25 milliliters of fresh DMEM medium with 20 millimolar HEPES and without sodium butyrate. After incubation for 48 hours, carefully collect the virus-containing medium with a pipette. To generate stable cell lines, start by seeding HCT-116 colon cancer cells and MDA-MB-231 breast cancer cells into a 12-well plate and 50, 000 cells per well.Incubate at 37 degrees Celsius and 5%carbon dioxide. When the cells are 60-70%confluent, wash the cells with PBS and add one milliliter of virus-containing medium to each well. The next day, carefully remove the virus-containing medium by aspiration.Then after washing the cells with PBS, add medium containing tetracycline-free FBS. After 72 hours, wash the cells as before and add medium supplemented with one microgram per milliliter puromycin for the selection of virus-transfected cells. Select virus-transduced cells by culturing cells in the presence of puromycin for three to 14 days.Observe partial killing of the wells by puromycin in the cells with virus-transduced cells. Non-transduced cells will not grow in the presence of puromycin. When these control cells have died, continue culture of the conditionally regulated cell lines for two weeks in the presence of antibiotics to create the conditionally regulated cell lines.Verify the efficiency of the conditional knockdown in the puromycin-resistant HCT-116 colon cancer and MDA-MB-231 breast cancer cells by adding medium with different concentrations of doxycycline and culturing the cells for 72 hours. After preparing cell lysate, verify the level of knockdown by western blot analysis. Levels of PAI-1 are shown here.To isolate peripheral blood monocytes, first prepare three 50 milliliter tubes containing 15 milliliters of density gradient solution. Then use a pipette controller set on gravitational flow to slowly layer 30 milliliters of diluted blood over the density gradient solution. Centrifuge in a swing rotor at 400 g at room temperature without breaks for 25 minutes.Following the centrifugation, dispose of the top layer and transfer the middle layer containing peripheral blood mononuclear cells to a fresh 50 milliliter tube and add FBS supplemented PBS up to 50 milliliters. Centrifuge at 120 g's at room temperature for 10 minutes. After the spin, remove the containing platelet-containing supernatant.Following the final centrifugation and collection, resuspend the pellet in 50 milliliters of FBS supplemented PBS and count the peripheral blood mononuclear cells using a hemocytometer. After centrifuging the cells at 120 g's, resuspend the pellet in FBS supplemented PBS containing one millimolar EDTA to a final concentration of 50 million cells per milliliter. Then begin to enrich for monocytes by adding 50 microliters of negative selection antibody cocktail for each milliliter of cell suspension and mix.Incubate for 10 minutes at room temperature. After the incubation, add 50 microliters of magnetic beads per milliliter, mix and incubate for another 10 minutes. Then add PBS with FBS and EDTA up to 25 milliliters and place the peripheral blood mononuclear mixture in a magnet that can accommodate a 50 milliliter tube.After incubating for 10 minutes at room temperature, the magnetic beads will adhere to the walls of the tube and sequester the non-monocytic cells from the solution. Use a 25 milliliter pipette to remove solution containing monocytes from the tube while being careful not to touch the sides of the tube with the pipette. After centrifuging and removing the supernatant, resuspend the pellet containing monocytes in RPMI medium containing 10%TET-free FBS and 1%pen-strep.Begin the assay by seeding 40, 000 HCT-116 or MDA-MB-231 cells in a 0.5 milliliter volume in the wells of a 24-well plate in triplicate. The next day, add a filter to the well. Then plate 8, 000 monocytes in a 0.3 milliliter volume onto a BSA-treated porous membrane insert placed on the top of the cancer cells and incubate at 37 degrees Celsius and 5%carbon dioxide.Collect the inserts after 48 hours. Shake off the excess medium and precisely swipe the inner surface with a cotton-tipped applicator to remove cells that did not migrate. Stain the outer side of the insert using the Wright-Giemsa method.After mounting the filters with the migrated macrophages side up, image the slides using a light microscope with a 20 power objective. Take pictures of nine fields per filter and count migrated macrophages in all fields. The Boyden Chamber Assay was used to quantify the migration of monocytes towards cancer cells.In the presence of MDA-MB-231 breast cancer cells, the migration of monocytes was inhibited by 33%after addition of doxycycline to the co-culture to down regulate PAI-1. This inhibition of monocyte migration was more pronounced in the presence of HCT-116 colon cancer cells where knockdown of PAI-1 by doxycycline administration reduced monocyte migration by 74%Similar results were observed when conditions medium for doxycycline-treated cancer cells was placed in the bottom of the wells as a source of chemoattractants. After watching this video, you should have a good understanding on how to use a functional tet-on system for gene expression knockdown in human cancer cell lines to study the chemoattractant effect of cancer-derived secreted protein PAI-1 on the human monocytes.

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

Research

JoVE Journal

Cancer Research

Lung Tumor Cell Recruitment Assay

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

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

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

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

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

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

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

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

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

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

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

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

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

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

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

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

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

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

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

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

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

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

Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal

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

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

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

Analysis of Side Population in Solid Tumor Cell Lines

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

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

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

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

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

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

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

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...