This work was supported by grants from the Canadian Institute of Health Research (CIHR#119325, 148629) and the Canadian Breast Cancer Foundation (CBCF) to XY. TA is supported by the Vanier Canada Graduate Scholarship and the Ontario International Graduate Scholarship. HJJVR is supported by a Queen Elizabeth II Graduate Scholarship in Science and Technology.

1. Investigation of a Putative Regulator of Hippo Signaling Using the LATS-BS
<ol>
	<li>Plating and transfection of cells
	<ol>
		<li>Preparation of cell culture
		<ol>
			<li>Warm 1x PBS, DMEM containing 10% FBS and 1% penicillin/streptomycin, and 0.25% trypsin-EDTA to 37 &#176;C in a water bath for approximately 30 min.</li>
			<li>Thoroughly clean a biosafety cabinet with 70% ethanol.</li>
			<li>Place tissue culture dishes, Pasteur pipettes, serological pipettes, and pipette tips into the tissue culture hood as required.</li>
			<li>Take out 1x PBS, the media, and the trypsin-EDTA from the water bath, clean them with 70% ethanol, and place them in the hood.</li>
		</ol>
		</li>
		<li>Maintenance and plating of the cells for transfection
		<ol>
			<li>Choose a cell line with a high transfection efficiency (e.g., HEK293) for the experiment. Check for the expression of Hippo pathway components in this cell line by western blot to make sure Hippo signaling is active.</li>
			<li>Grow HEK293 cells in DMEM in 10 cm Petri dishes in an incubator at 37 &#176;C and 5% CO2. Monitor the cells under a microscope every day. Passage the cells as described below when 80&#8211;90% confluency is observed.</li>
			<li>Wash the cells by adding 5 mL of 1x PBS into a plate, gently swirling the plate, and aspirating the media completely.</li>
			<li>Add 1 mL of trypsin-EDTA to the plate. Swirl the plate to ensure that the trypsin evenly covers the cells.</li>
			<li>Incubate the cells at 37 &#176;C in a CO2 incubator for 5 min or until all the cells are detached.</li>
			<li>Add 4 mL of media (with 10% FBS and 1% penicillin/streptomycin) to neutralize the trypsin, resuspend the cells by pipetting up and down several times, and dispense one-tenth (0.5 mL) of the cells into new 100 mm plates (for a passing ratio of 1:10) containing 9.5 mL of complete media.</li>
			<li>For LATS-BS transfection, count the cells using a hemocytometer and plate 2 x 105 cells into each well of a 12-well plate 24 h before transfection. 
			NOTE: Using higher cell numbers may increase the background bioluminescent signal as Hippo signaling activity is regulated by cell confluency. Using lower cell numbers may increase cell sensitivity to transfection reagents and increase cell death24.</li>
		</ol>
		</li>
		<li>Transfection of the LATS-BS alone or together with the candidate gene
		<ol>
			<li>Miniprep LATS-BS plasmids (NLuc-YAP15-pcDNA3.1-Hygro and 14-3-3-CLuc-pcDNA3.1-Hygro) fresh from DH5a bacteria liquid culture (necessary to achieve an optimum LATS-BS expression).</li>
			<li>1 h before transfection, aspirate the medium from the plate, add 500 &#181;L of complete growth medium to each well of the 12-well plates and return the cells to the incubator. 
			NOTE: The following protocol describes transfection methods using a commercially available polymer-based transfection reagent. Other transfection reagents may also be suitable for transfection; however, we have not yet tested them.</li>
			<li>Make solutions A and B as described in Table 1. For N transfections, make (N + 0.5) x solution B premix.</li>
			<li>Immediately add the diluted polymer-based transfection reagent (solution B) to DNA (solution A). Pipette up and down gently to mix.</li>
			<li>Incubate the sample at room temperature for 15 min to allow transfection complexes to form.</li>
			<li>Add the total volume (76 &#181;L) of transfection complexes dropwise to each well of the 12-well plate with gentle swirling.</li>
			<li>Incubate the cells overnight (18&#8211;24 h) at 37 &#176;C. 
			Note: For cells that are sensitive to polymer-based transfection reagents, the incubation time can be reduced to 5 h.</li>
			<li>The day after transfection, remove the transfection-complex-containing media and replace it with fresh complete media. Culture the cells at 37 &#176;C for another day to perform luciferase assay.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Luciferase assay 
	NOTE: The Luciferase Reporter Assay System detecting firefly and Renilla together in one assay is used for the luciferase assays. If Renilla was not used as an internal control, perform one-step luciferase assay by adding LARII without adding the Renilla detection reagent.
	<ol>
		<li>Preparation of the cell lysates
		<ol>
			<li>Dilute 5x passive lysis buffer (PLB) with distilled water as follows:</li>
			<li>Total volume of 1x PLB required = N x (50 &#956;L of 5x PLB + 200 &#956;L of dH2O) = N x 250 &#956;L of 1x PLB per well of the 12-well plates (N = number of samples).</li>
			<li>Aspirate the media completely. Add 1 mL of 1x PBS into each well. Swirl the plate gently to remove detached cells and residual growth media. Aspirate the 1x PBS completely. Make sure no residual 1x PBS remains prior to the addition of 1x PLB.</li>
			<li>Add 250 &#956;L of 1x PLB/well.</li>
			<li>Place the culture plates on a rocking platform with gentle rocking to ensure complete and even coverage of the cell monolayer with 1x PLB. Rock the culture plates at room temperature for 15 min to allow the cells to lyse.</li>
			<li>Transfer the lysate to a 1.5 mL microcentrifuge tube.</li>
		</ol>
		</li>
		<li>Measuring the bioluminescent signal
		<ol>
			<li>Take out the LARII (20 &#956;L/sample) from -80 &#176;C and equilibrate it to room temperature.</li>
			<li>Prepare Renilla detection reagent for N assays: add (N + 1) x 0.4 &#956;L of 50x Substrate to (N + 1) x 19.6 &#956;L of its Buffer.</li>
			<li>Predispense 10 &#956;L of each cell lysate to 1.5-mL tubes.</li>
			<li>Program a luminometer to perform a 2-s premeasurement delay, followed by a 10-s measurement period for each dual luciferase assay. 
			NOTE: The first and second measurements will be corresponding to the LATS-BS signal and the Renilla internal control signal, respectively. Any luminometer compatible with a single or dual luciferase can be used.</li>
			<li>Carefully transfer 20 &#956;L of LARII reagent into one tube and quickly pipette up and down 3x to mix it.</li>
			<li>Immediately place the tube in the luminometer and initiate the reading.</li>
			<li>Remove the sample tube from the luminometer, immediately add 20 &#956;L of Renilla Reagent, and mix by pipetting up and down 3x. Bring the sample in the luminometer and initiate the next reading. Record the measurements.</li>
			<li>Discard the reaction tube and proceed to the next sample.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. A screen to Identify Hippo Pathway Regulators Using the LATS-BS
<ol>
	<li>Cell culture and transfections
	<ol>
		<li>Culture HEK293AD cells as described in section 1.1. 
		NOTE: HEK293AD is more adherent than other HEK293 cell lines, which makes it a good cell line for use in screens.</li>
		<li>Detach cells as described in section 1.1.2. Count and plate 2 x 106 cells in 100 mm plates 24 h before transfection.</li>
		<li>1 h before transfection, aspirate the media from the plate and replace them with 5 mL of fresh complete growth media.</li>
		<li>Make solutions A and B as described in Table 2.</li>
		<li>Immediately add the diluted polymer-based transfection reagent (solution B) to DNA (solution A). Pipette up and down to mix.</li>
		<li>Incubate the sample at room temperature for 15 min to allow transfection complexes to form.
		<ol>
			<li>Add the total volume (500 &#181;L) of transfection complexes dropwise to cells with gentle swirling.</li>
			<li>Incubate the cells overnight at 37 &#176;C.</li>
			<li>The next day, trypsinize and count the cells. Plate 1 x 104 to 2 x 104 cells into each well of a 96-well plate in a total volume of 100 &#181;L of media per well. Incubate for another day.</li>
		</ol>
		</li>
		<li>The next day, add agents/small molecules regulating the Hippo pathway at a final concentration of 10 &#181;M to each well, 1&#8211;4 h before performing the luciferase assay. 
		Note: For most small molecules/agents, a 4 h treatment at 10 &#181;M does not affect cell viability. If a lower concentration is used, treatment may be extended for a longer period of time.</li>
	</ol>
	</li>
	<li>Luciferase assay
	<ol>
		<li>In vitro luciferase assay
		<ol>
			<li>Aspirate the media from the cells completely. Wash the cells with 100 &#181;L of 1x PBS in each well. Aspirate completely.</li>
			<li>Add 20 &#181;L of 1x PLB (prepared as described in step 1.2.1.1) into each well of the 96-well plates.</li>
			<li>Place the culture plates on a rocking platform with gentle rocking at room temperature for 15 min.</li>
			<li>Carefully transfer 20 &#181;L of LARII reagent into each well and pipette up and down 3x to mix.</li>
			<li>Immediately place the plate in a plate-reading luminometer and initiate the reading.</li>
		</ol>
		</li>
		<li>Living cell luciferase assay
		<ol>
			<li>Make 11x D-luciferin stock as follows: dissolve 1.5 mg of D-luciferin powder in 1 mL of normal culture media.</li>
			<li>Transfer 10 &#181;L of 11x D-luciferin into each well and mix it gently with the media which is already in the well.</li>
			<li>Replace the cells into the incubator for 10 min.</li>
			<li>Place the plate in the luminometer and initiate the reading. 
			Note: If the signal intensity is not strong enough at this concentration of D-luciferin, another 10 &#181;L of the D-luciferin stock may be added to the cells.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Justice, R. W., Zilian, O., Woods, D. F., Noll, M., Bryant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+tumor+suppressor+gene+warts+encodes+a+homolog+of+human+myotonic+dystrophy+kinase+and+is+required+for+the+control+of+cell+shape+and+proliferation.">The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation.</a> Genes &amp; Development. 9 (5), 534-546 (1995).</li><li>Xu, T., Wang, W., Zhang, S., Stewart, R. A., Yu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+tumor+suppressors+in+genetic+mosaics:+the+Drosophila+lats+gene+encodes+a+putative+protein+kinase.">Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase.</a> Development. 121 (4), 1053-1063 (1995).</li><li>Taha, Z., Janse van Rensburg, H. J., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+Pathway:+Immunity+and+Cancer.">The Hippo Pathway: Immunity and Cancer.</a> Cancers. 10 (4), 94 (2018).</li><li>Maugeri-Sacc&#224;, M., De Maria, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+pathway+in+normal+development+and+cancer.">The Hippo pathway in normal development and cancer.</a> Pharmacology &amp; Therapeutics. 186, 60-72 (2018).</li><li>van Rensburg, H. J. J., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+the+Hippo+pathway+in+cancer+metastasis.">The roles of the Hippo pathway in cancer metastasis.</a> Cellular Signalling. 28 (11), 1761-1772 (2016).</li><li>Yeung, B., Yu, J., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+Hippo+pathway+in+lung+development+and+tumorigenesis.">Roles of the Hippo pathway in lung development and tumorigenesis.</a> International Journal of Cancer. 138 (3), 533-539 (2016).</li><li>Zhao, Y., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+pathway+in+chemotherapeutic+drug+resistance.">The Hippo pathway in chemotherapeutic drug resistance.</a> International Journal of Cancer. 137 (12), 2767-2773 (2015).</li><li>Yu, F. -. X., Guan, K. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+pathway:+regulators+and+regulations.">The Hippo pathway: regulators and regulations.</a> Genes &amp; Development. 27 (4), 355-371 (2013).</li><li>Yang, X., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+size+control+in+development+and+human+diseases.">Molecular mechanism of size control in development and human diseases.</a> Cell Research. 21 (5), 715 (2011).</li><li>Visser, S., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LATS+tumor+suppressor:+a+new+governor+of+cellular+homeostasis.">LATS tumor suppressor: a new governor of cellular homeostasis.</a> Cell Cycle. 9 (19), 3892-3903 (2010).</li><li>Zhao, B., 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=Inactivation+of+YAP+oncoprotein+by+the+Hippo+pathway+is+involved+in+cell+contact+inhibition+and+tissue+growth+control.">Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.</a> Genes &amp; Development. 21 (21), 2747-2761 (2007).</li><li>Hao, Y., Chun, A., Cheung, K., Rashidi, B., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppressor+LATS1+is+a+negative+regulator+of+oncogene+YAP.">Tumor suppressor LATS1 is a negative regulator of oncogene YAP.</a> Journal of Biological Chemistry. 283 (9), 5496-5509 (2008).</li><li>Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., Umezawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+Luciferase+as+an+Optical+Probe+for+Detecting+Protein.">Split Luciferase as an Optical Probe for Detecting Protein.</a> Protein Interactions in Mammalian Cells Based on Protein Splicing. Analytical Chemistry. 73 (11), 2516-2521 (2001).</li><li>Hashimoto, T., Adams, K. W., Fan, Z., McLean, P. J., Hyman, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+oligomer+formation+of+amyloid-&#946;+peptide+using+a+split-luciferase+complementation+assay.">Characterization of oligomer formation of amyloid-&#946; peptide using a split-luciferase complementation assay.</a> Journal of Biological Chemistry. 286 (31), 27081-27091 (2011).</li><li>Decock, 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=Analysis+by+a+highly+sensitive+split+luciferase+assay+of+the+regions+involved+in+APP+dimerization+and+its+impact+on+processing.">Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.</a> FEBS Open Bio. 5 (1), 763-773 (2015).</li><li>Azad, T., Tashakor, A., Rahmati, F., Hemmati, R., Hosseinkhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillation+of+apoptosome+formation+through+assembly+of+truncated+Apaf-1.">Oscillation of apoptosome formation through assembly of truncated Apaf-1.</a> European Journal of Pharmacology. 760, 64-71 (2015).</li><li>Br&#252;ckner, A., Polge, C., Lentze, N., Auerbach, D., Schlattner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+two-hybrid,+a+powerful+tool+for+systems+biology.">Yeast two-hybrid, a powerful tool for systems biology.</a> International Journal of Molecular Sciences. 10 (6), 2763-2788 (2009).</li><li>Pattnaik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+plasmon+resonance.">Surface plasmon resonance.</a> Applied Biochemistry and Biotechnology. 126 (2), 79-92 (2005).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer.">Fluorescence resonance energy transfer.</a> Current Opinion in Biotechnology. 6 (1), 103-110 (1995).</li><li>Azad, T., Tashakor, A., Hosseinkhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-luciferase+complementary+assay:+applications,+recent+developments,+and+future+perspectives.">Split-luciferase complementary assay: applications, recent developments, and future perspectives.</a> Analytical and Bioanalytical Chemistry. 406 (23), 5541-5560 (2014).</li><li>Hu, C. -. D., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+visualization+of+multiple+protein+interactions+in+living+cells+using+multicolor+fluorescence+complementation+analysis.">Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.</a> Nature Biotechnology. 21 (5), 539-545 (2003).</li><li>Hosseinkhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+enigma+of+multicolor+bioluminescence+of+firefly+luciferase.">Molecular enigma of multicolor bioluminescence of firefly luciferase.</a> Cellular and Molecular Life Sciences. 68 (7), 1167-1182 (2011).</li><li>Paulmurugan, R., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+library+screening+for+developing+an+improved+split-firefly+luciferase+fragment-assisted+complementation+system+for+studying+protein-protein+interactions.">Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions.</a> Analytical Chemistry. 79 (6), 2346-2353 (2007).</li><li>Azad, 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=A+LATS+biosensor+screen+identifies+VEGFR+as+a+regulator+of+the+Hippo+pathway+in+angiogenesis.">A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis.</a> Nature Communications. 9 (1), 1061 (2018).</li><li>Moroishi, 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=A+YAP/TAZ-induced+feedback+mechanism+regulates+Hippo+pathway+homeostasis.">A YAP/TAZ-induced feedback mechanism regulates Hippo pathway homeostasis.</a> Genes &amp; Development. 29 (12), 1271-1284 (2015).</li></ol>

The authors have nothing to disclose.

While the Hippo pathway plays critical roles in various biological processes, and dysregulation of the Hippo pathway leads to cancer6, how the Hippo pathway is regulated in response to various stimuli is not completely understood. In addition, there has been no quantitative and real-time system to assess the activity of core Hippo components. Recently, we developed and validated a novel biosensor for measuring LATS kinase activity in the Hippo pathway24. This biosensor can be used not only for quantitatively and accurately monitoring Hippo signaling activity in living cells but also for screens of drugs targeting the Hippo pathway for cancer therapy. In addition, the biosensor-based screening can lead to the discovery of novel genes responsible for the regulation of the Hippo pathway. Given the importance of Hippo signaling in cancer, finding crucial crosstalk between other genes and the Hippo pathway would be a valuable study to undertake for all cancer biologists. Therefore, this biosensor has the potential to make innovations in the current treatment of cancer and might provide a useful new therapeutic strategy for successful treatments of cancer.
There are several important points that should be considered when using the LATS-BS. First, in our experience, the LATS-BS has the greatest sensitivity when the plasmids used for transfection are extracted fresh from bacteria. Second, the amount&#160;of LATS-BS plasmids transfected into cells will affect the LATS-BS sensitivity and signal intensity. The amount of plasmid DNA can be increased to 400 ng/fragment for the LATS-BS and to 600 ng for the gene of interest per well of a 12-well plate. In general, using more plasmid will give a higher signal intensity but less sensitivity. Third, cell confluency can affect the LATS-BS signal background. Consistent with the well-established role of Hippo signaling in cellular responses to confluency, we have observed that cells with higher confluency have a higher background LATS-BS signal due to the activation of endogenous LATS. Accordingly, sparsely-plated cells show a lower LATS-BS background signal. Optimal cell confluency can be selected depending on the purpose of the experiment (high confluency for observing LATS inhibition; low confluency for observing LATS activation). Fourth, Renilla may be used as an internal control for transfection efficiency, and for hard-to-transfect cells (e.g., MCF10A), the transfection reagent:DNA ratio can be modified to 4:1. Finally, we strongly recommend cotransfecting MST2 as a positive control and YAP-S127A as a negative control each time the biosensor is used.
Every tool has its limitations. The LATS-BS is the artificial substrate of an endogenous protein, which can always outcompete the true substrate of the protein of interest. This effect is known as a buffering effect of the biosensor. Improving the biosensor with higher signal intensity and lower expression levels may help to overcome this problem. Another limitation is that feedback pathways may complicate the interpretation of data from the LATS-BS. There are many linear and non-linear interacting signaling pathways that likely influence the LATS-BS signal. The LATS-BS only demonstrates the cumulative effect of a stimulus on LATS kinase activity. It should be noted, however, that that limitation is not unique to the LATS-BS but rather is commonly encountered in studies of molecular biology. Thus, LATS-BS data should be interpreted in the context of multiple probative experiments.

The Hippo signaling pathway was first identified in Drosophila as a novel regulator of cell growth and animal size1,2. Since its initial discovery, mounting evidence has convincingly shown that the Hippo pathway plays critical roles in the development (e.g., early embryonic development, organ size control, and three-dimensional [3-D] morphology), tumorigenesis (e.g., tumor development, metastasis, angiogenesis, immune evasion, genomic instability, stress response, and drug resistance), and tissue homeostasis (e.g., stem cell renewal and differentiation and tissue regeneration after injury)3,4,5,6,7,8,9,10. Hippo signaling is frequently dysregulated in various cancers7,8,9,10,11,12. Therefore, elucidating functions of the Hippo pathway in cancer biology and therapeutics and regenerative medicine has become one of the hottest areas in biomedical research.
In brief, in the Hippo pathway, upon activation by upstream regulators (e.g., cell-cell contact, nutrient stress, and extracellular matrix [ECM]), MST1/2 (MST; mammalian homologs of Drosophila Hippo) serine/threonine (S/T) kinases phosphorylate/activate adaptor proteins hMOB1 and WW45, as well as LATS1/2 (LATS) kinases which, subsequently, phosphorylate transcriptional co-activator YAP and its paralog TAZ at conserved HX(H/R/K)XX(S/T) (H, histidine; R, arginine; K, lysine; S, serine; T, threonine; X, any amino acids) motifs, including YAP-S127 and TAZ-S8911,12. S127-phosphorylated YAP (YAP-pS127) and S89-phosphorylated TAZ (TAZ-pS89) are degraded by ubiquitination or bind to cytoplasmic protein 14-3-3 and are prevented from interacting with TEAD1-4 transcription factors in the nucleus to transactivate downstream genes involved in the cell proliferation and apoptosis (Figure 1). Despite the tremendous interest in the Hippo pathway, few tools for measuring Hippo signaling exist and those that do have been historically limited to reporters of YAP/TAZ/TEAD transcriptional output. Indeed, until very recently, there were no tools for measuring the dynamics and activity of the Hippo signaling components in a quantitative, real-time, high-throughput, and non-invasive manner both in vitro and in vivo.
Given our emerging understanding of the role of protein-protein interactions in physiology and pathology, there is great interest in the development of tools that can be used to study these interactions in a quantitative and real-time manner13,14,15,16. Indeed, there has been significant progress in the development of bio-analytical strategies, including the yeast two-hybrid (Y2H)17, the surface plasmon resonance (SPR)18, and F&#246;rster resonance energy transfer (FRET)19 assays, to evaluate protein-protein interactions. However, these approaches carry the limitation of requiring significant optimization of the reporter orientation, such that many constructs must be tested to find an efficient one. Further, these approaches also have a relatively low signal-to-noise ratio, such that discerning a true positive signaling can be challenging.
Protein complementation assays were developed to overcome these limitations. The first generation of protein complementation assays was based on split multicolor fluorescence proteins and could not solve the aforementioned limitations20. Multicolor fluorescence proteins consist of only one domain, making it difficult to split them into two separate stable fragments with low affinity and background noise21. Subsequently, firefly luciferase was identified as a new candidate for use in developing split protein complementation assays. In this approach, firefly luciferase is split into two fragments (N-terminal and C-terminal luciferase [NLuc and CLuc]) with each fragment fused to a target protein of interest. If the NLuc and CLuc are brought into proximity upon the interaction of the two target proteins, luciferase activity is reconstituted and bioluminescent light is generated in the presence of luciferin substrate and ATP22. In 2001, by performing a combinatorial screening using a library containing NLuc and CLuc fragments cut at various sites and attached to proteins with different linkers, Paulmurugan and Gambhir at Stanford University developed an optimized split-firefly luciferase fragment-assisted complementation system for protein-protein interactions23. In this system, firefly luciferase is cut at amino acid (aa) 398 to form NLuc and CLuc, which are attached to two proteins of interest using a flexible linker of eight glycine residues and two serine residues.
Using a similar approach, we recently developed a new LATS-BS by fusing NLuc to 15 aa of YAP surrounding the LATS phosphorylation site at S127 (YAP15) and CLuc with 14-3-3. The full-length YAP protein was not used, to avoid confounding signals by post-translational modifications of YAP (e.g., phosphorylation at other sites and ubiquitination) by other upstream regulators. The LATS-BS presented here can non-invasively monitor Hippo signaling activity both in vitro in living cells and in vivo in mice20,24 (Figure 2). Here, we describe a detailed protocol for measuring LATS kinase activity in vitro using the LATS-BS. First, we show how the LATS-BS can be used to investigate the effect of an overexpressed protein on LATS activity. Then, we show how the biosensor can be used to monitor the activity of the Hippo pathway after treatment with agents regulating the Hippo pathway. This protocol could be used to identify and characterize signaling pathways or stimuli regulating LATS kinase activity.

<table><tbody><tr><td>&lt;strong&gt;Trypsin-EDTA&amp;nbsp;&lt;/strong&gt;</td><td>Life Technologies</td><td>2520056</td><td>0.25%</td></tr><tr><td>&lt;strong&gt;Fetal Bovine Serum (FBS)&lt;/strong&gt;</td><td>Sigma</td><td>F1051</td><td></td></tr><tr><td>&lt;strong&gt;DMEM&lt;/strong&gt;</td><td>Sigma</td><td>D6429-500ml</td><td>DMEM with high glucose&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Penicillin Streptomycin&lt;/strong&gt;</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>&lt;strong&gt;PolyJet &lt;em&gt;In Vitro&lt;/em&gt; DNA Transfection Reagent&amp;nbsp;&lt;/strong&gt;</td><td>Signagen</td><td>SL100688.5</td><td></td></tr><tr><td>&lt;strong&gt;5x Passive lysis buffer&lt;/strong&gt;</td><td>Promega</td><td>E194A</td><td>30 ml</td></tr><tr><td>&lt;strong&gt;Dual-Glo&amp;reg; Luciferase Assay System&lt;/strong&gt;</td><td>Promega</td><td>E2940</td><td>100 ml kit</td></tr><tr><td>&lt;strong&gt;20/20 luminometer&lt;/strong&gt;</td><td>Turner Biosystems</td><td>998-2036</td><td>Single tube reader luminometer</td></tr><tr><td>&lt;strong&gt;GloMax&amp;reg;Navigator with dual injectors&lt;/strong&gt;</td><td>Promega</td><td>GM2010</td><td>96-well plates reader luminometer</td></tr></tbody></table>

monitoring hippo signaling pathway activity using a luciferase-based large tumor suppressor (lats) biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

The LATS-BS was cotransfected with different genes to evaluate their effect on LATS activity (Figure 3). In this experiment, Renilla was used as an internal control. The transient expression of YAP-5SA, a constitutive active form of YAP, causes increasing levels of YAP transcriptional targets and a subsequent increase in LATS kinase activity through an established feedback pathway25. MST, the upstream activator of LATS in the Hippo pathway (Figure 1), also increases the biosensor signal intensity. However, VEGFR overexpression, which triggers PI3K/MAPK signaling, inhibits LATS and decreases the signal intensity of the LATS-BS.
In the second protocol, LATS-BS was transfected into HEK293AD and, 24 hours after the transfection, cells were passed into 96-well plates. 40 h after the transfection, the cells were treated with different known small molecule regulators of Hippo signaling followed by a luciferase assay (Figure 4). VEGFR, PI 3-kinase, and cellular energy level are three known negative regulators of the LATS kinase24. In this experiment, GDS0941, a PI 3-kinase inhibitor, axitinib and SU4312, inhibitors of VEGFR, and 2-D glucose, which causes energy stress by an inhibition of glucose metabolism and ATP production, were used to activate the LATS-BS. Inversely, LPA, S1P, and TPA were used to inhibit the LATS-BS. This experiment shows that the LATS-BS can be used to measure both the activation and the inhibition effect of a small molecule on the LATS kinase activity.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution A</td>
			<td></td>
		</tr>
		<tr>
			<td>pCDNA3.1-Hygro-NLuc-YAP15</td>
			<td>1 &#956;L (50 ng)</td>
		</tr>
		<tr>
			<td>pCDNA3.1-Hygro-14-3-3-CLuc</td>
			<td>1 &#956;L (50 ng)</td>
		</tr>
		<tr>
			<td>Plasmid expressing a candidate gene (e.g. MST)&#160;</td>
			<td>1 &#956;L (400 ng)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; or empty vector (e.g. pcDNA3.1-Hygro)&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla vector (pRL-TK) (optional)&#160;&#160;</td>
			<td>1 &#956;L (20 ng)</td>
		</tr>
		<tr>
			<td>DMEM (no serum/antibiotics)&#160;&#160;</td>
			<td>34 &#956;L</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>38 &#956;L</td>
		</tr>
		<tr>
			<td>Solution B</td>
			<td></td>
		</tr>
		<tr>
			<td>Polymer-based transfection reagent</td>
			<td>1.5 &#956;L</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; (3:1 ratio with total &#956;g DNA)</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (no serum/antibiotics)&#160;&#160;</td>
			<td>36.5 &#956;L</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>38 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 1: Protocol for making solutions A and B using a polymer-based transfection reagent for investigating a putative regulator of the Hippo pathway with the LATS-BS.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution A</td>
			<td></td>
		</tr>
		<tr>
			<td>pCDNA3.1-Hygro-NLuc-YAP15</td>
			<td>10 &#956;L (3 &#956;g)</td>
		</tr>
		<tr>
			<td>pCDNA3.1-Hygro-14-3-3-CLuc</td>
			<td>10 &#956;L (3 &#956;g)</td>
		</tr>
		<tr>
			<td>DMEM (no serum/antibiotics)&#160;&#160;</td>
			<td>230 &#956;L</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>250 &#956;L</td>
		</tr>
		<tr>
			<td>Solution B</td>
			<td></td>
		</tr>
		<tr>
			<td>Polymer-based transfection reagent</td>
			<td>18 &#956;L</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; (3:1 ratio with total &#956;g DNA)</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (no serum/antibiotics)&#160;&#160;</td>
			<td>232 &#956;L</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>250 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 2: Protocol for making solutions A and B using a polymer-based transfection reagent for screening to identify regulators of the Hippo pathway using the LATS-BS.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58416/58416fig1.jpg" /> 
Figure 1: The Hippo pathway in mammals. The Hippo pathway restricts organ size by phosphorylating and inhibiting YAP and TAZ. When the Hippo pathway is activated, SAV, LATS1/2, and MOB are phosphorylated and activated by MST1/2. Activated LATS1/2 phosphorylates YAP/TAZ at conserved serine-containing sites. 14-3-3 interacts with phosphorylated YAP/TAZ, thereby inhibiting their nuclear localization and co-transactivation of downstream gene targets through TEADs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58416/58416fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58416/58416fig2.jpg" /> 
Figure 2: LATS-BS split-protein complementation assay based on luciferase. Luciferase N- and C-terminus domains (NLuc and CLuc) are attached to 15 amino acid surrounding YAP-S127 and 14-3-3, respectively. YAP15/14-3-3 binding after LATS phosphorylation promotes luciferase complementation and bioluminescence signal emission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58416/58416fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58416/58416fig3v2.jpg" /> 
Figure 3: Results from a representative experiment evaluating the effect of a gene on LATS activity. The biosensor can be expressed alone or together with other genes in HEK293AD cells to investigate their effect on LATS kinase activity (mean &#177; SD; n = 3, **p &#60;0.01, ***p &#60;0.001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58416/58416fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58416/58416fig4.jpg" /> 
Figure 4: Results from a representative experiment evaluating the effect of small molecules on LATS activity in HEK293AD cells. HEK293AD cells were transfected with the LATS-BS and treated with the following drugs: PI3K inhibitor (GDC0941), 10 &#956;M for 4 h; VEGFR inhibitor (axitinib), 10 &#956;M for 4 h; 2-deoxyglucose, 25 mM for 1 h; LPA, 10 &#956;M for 1 h; sphingosine-1-phosphophate (S1P), 1 &#956;M for 1 h; 12-O-tetradecanoylphorbol-13-acetate (TPA), 5 nM for 1 h; VEGFR inhibitor (SU4312), 10 &#956;M for 4 h (mean &#177; SD; n = 3, **p &#60;0.01, ***p &#60;0.001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58416/58416fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor at JoVE.com

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

monitoring-hippo-signaling-pathway-activity-using-luciferase-based

Nanyang Technological University

Research

JoVE Journal

Cancer Research

19.5K Views.  Queen's University. Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

Video: Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Neuroscience

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Medicine

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

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  ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Developmental Biology

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

Summary

Explore More Videos

Chapters in this video

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

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

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells