We acknowledge funding support from CPRIT (RP100119) and the Welch Foundation (I-1665).

Entire protocol takes approximately 4 days.
1. Preparation of Cells for siRNA and Reporter Transfection
We will introduce here a protocol for interrogating siRNA libraries using a small set of siRNAs (384 different siRNA pools array in 96 well format with each pool consisting of 4x siRNAs targeting a single gene) from a genome-wide siRNA library for illustrative purposes. For this particular study, we will exploit HCT116 cells that exhibit deviant Wnt and Kras signaling, and p53 activity. The appropriate cell line in studies aimed at interrogating other cellular processes of interest will have to be identified and evaluated in a similar fashion.
<ol>
	<li>Wash 5 x 10 cm2 plates of 80-90% confluent HCT116 cells with PBS then harvest cells by trypsinization using 1 ml of trypsinization solution/plate followed by neutralization with 10 ml of Dulbecco's modified eagle medium (DMEM) containing 2% fetal bovine serum (FBS) and 1% penicillin/streptomycin/plate.</li>
	<li>Transfer suspended cells to a 50 ml conical tube and centrifugate at ~130 x g for 5 min, remove supernatant, and re-suspend cell pellet in 10 ml of DMEM/ 2%FBS/ 1% penicillin/streptomycin.</li>
	<li>Count the cell number with a cell counter, and then make a cell solution with 105 cells/ml, keep 150 ml cell suspension for the next step. Plate 104 cells (100 &mu;l) in each well of a 96 well cell culture plate using a Multidrop automated liquid dispenser (henceforth Multidrop). The cell suspension should be sufficient in this case for plating 12 x 96 well plates.</li>
	<li>Incubate the plates with cells at 37 &deg;C in an incubator with 5% CO2 while preparing the transfection mixture.</li>
</ol>
2. Preparing the Reporter Construct Stock Solution
<ol>
	<li>Isolate high-quality reporter construct DNA using standard midiprep kits (NucleoBond Xtra Midi produced by Clontech) and dilute the DNA to a final concentration of 1 mg/ml for each reporter.</li>
	<li>Prepare 100 &mu;l of reporter construct stock solution by combining 30 &mu;l of p53-FL, 30 &mu;l of 8X TCF-RL, 30 &mu;l of Elk1-Gal4, 6 &mu;l UAS-CL and 4 &mu;l of H2O to achieve a DNA mix with a final 1:1:1:0.2 ratio of the reporters. Final DNA concentration of this DNA mix stock solution is 0.3 mg/ml.</li>
</ol>
3. Preparing the siRNA/DNA Transfection Mix
In this part of the protocol, we will prepare siRNA/DNA transfection mixes that will be sufficient for transfecting 12 x 96 well plates. For this test library of 4 x 96 well plate of siRNAs, we will transfect each siRNA pool in triplicate. The transfection reagent we will use is called Effectene (Qiagen). In our hands this is the only reagent that works for the simultaneous delivery of siRNA and DNA into cultured cells.
<ol>
	<li>Dilute 80 &mu;l of reporter construct stock solution in 8 ml of EC buffer (Effectene Kit) to achieve a DNA solution with a final concentration of 3 &mu;g/ml. In general, prepare enough DNA solution mix for immediate use.</li>
	<li>Plate 20 &mu;l of this DNA solution to each well of a 96 well/plate. The number of 96 well plates will depend on how many siRNA pools will be tested. For example, in this case we will need 4 x 96 well plates to evaluate 384 different siRNA pools.</li>
	<li>The siRNAs to be tested should be at a stock concentration of 5 &mu;M. If not, they can be replica plated and diluted to this concentration using the recommended dilution buffer associated with each library.</li>
	<li>Transfer 2 &mu;l of each 5 &mu;M siRNA stock to the corresponding well of the PCR plate containing the diluted DNA stock with a Biomek Automated Liquid Handler. Depending on the scale of the study, a multi-channel pipettor may suffice.</li>
	<li>Now, we will add 1 &mu;l of Enhancer solution (Effectene kit) to each well of the DNA/siRNA plate. This again can be accomplished either with an automated liquid handler or multichannel pipettor.</li>
	<li>Allow the Enhancer reaction to occur at RT for 5 min then add 3 &mu;l of Effectene transfection reagent to each well and incubate at RT for an additional 10 min.</li>
	<li>To stop transfection complex formation, add 10 &mu;l of DMEM/ 2% FBS/ 1% penicillin/streptomycin to each well of the PCR plate.</li>
	<li>Transfer 10 &mu;l of the transfection mix to each well of the 96 well plate containing the cells (retrieved from the cell incubator). As each transfection will be performed in triplicate, the same mix will be applied to two additional 96 well plates containing cells.</li>
	<li>Incubate the cells with transfection mixtures added at 37 &deg;C in a humidified environment with 5% CO2 for 36 hr.</li>
</ol>
4. Determine Luciferase Activities from Cell Samples
After 36 hr, luciferase activities are ready for measurement in transfected cells. The endpoint should be determined on a per-experiment basis. In our study, a 36 hr incubation time had previously yielded a sufficiently robust signal for meaning outcome determination. As this study incorporates multiple reporters (one secreted into the culture medium, and two others expressed in the cell cytoplasm), we will obtain measurements from both culture medium as well as a cellular lysate.
<ol>
	<li>Detection of CL activity in the culture medium:
	<ol>
		<li>20 &mu;l of culture medium is replica-plated in a white opaque 96 well plate (either using the Biomek Liquid Handler or multichannel pipettor).</li>
		<li>Add 20 &mu;l of CL assay buffer (Targeting Systems) to each well.</li>
		<li>Add 10 &mu;l of Cypridina luciferin substrate (Targeting Systems) to each well and detect CL activity using a luminometer (the Pherastar plate reader produced by BMG for example).</li>
	</ol>
	</li>
	<li>Detection of FL and RL activity:
	<ol>
		<li>Lyse the cells by first removing the culture medium. This can be accomplished rapidly by turning plate upside down and tossing medium in a sink. Remaining medium can be removed by gently tapping the upside down plate on paper towels. Add 30 &mu;l of 1X Passive Lysis Buffer (Promega) to each well of cells using the Multidrop and place on a platform rocker (Bellco is one company that makes these) set a medium speed for 5 min at the RT.</li>
		<li>Add 20 &mu;l of the Luciferase Assay Reagent II (part of the Promega Dual Luciferase Kit) using the Multidrop and immediately measure FL activity using the luminometer. Next, add Stop &amp; Glo Reagent (Promega) that will quench the FL activity and allow measurement of RL activity. Note: unless you are using substrate mixes that allow extended signal duration (such as Dual-Glo Luciferase Kits from Promega), the use of flash kits requires rapid measurement upon substrate addition (within 5 min).</li>
	</ol>
	</li>
</ol>

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	<li>Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S., Kricka, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescent+and+bioluminescent+reporter+gene+assays.">Chemiluminescent and bioluminescent reporter gene assays.</a> Analytical Biochemistry. 219, 169-181 (1994).</li><li>Miraglia, L. J., King, F. J., Damoiseaux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+light:+luminescent+reporter+gene+assays.">Seeing the light: luminescent reporter gene assays.</a> Combinatorial Chemistry &amp; High Throughput Screening. 14, 648-657 (2011).</li><li>McGuire, S. E., Roman, G., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+systems+in+Drosophila:+a+synthesis+of+time+and+space.">Gene expression systems in Drosophila: a synthesis of time and space.</a> Trends in Genetics: TIG. 20, 384-391 (2004).</li><li>Smirnova, N. 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=Development+of+Neh2-luciferase+reporter+and+its+application+for+high+throughput+screening+and+real-time+monitoring+of+Nrf2+activators.">Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators.</a> Chem. Biol. 18, 752-765 (2011).</li><li>Chen, 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=Small+molecule-mediated+disruption+of+Wnt-dependent+signaling+in+tissue+regeneration+and+cancer.">Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.</a> Nat. Chem. Biol. 5, 100-107 (2009).</li><li>Tang, 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=A+genome-wide+RNAi+screen+for+Wnt/beta-catenin+pathway+components+identifies+unexpected+roles+for+TCF+transcription+factors+in+cancer.">A genome-wide RNAi screen for Wnt/beta-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9697-9702 (2008).</li><li>Lum, L. D., Kulak, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Luciferase+Reporter+Assay+Systems.">Multiplexed Luciferase Reporter Assay Systems.</a> United States patent. , (2012).</li><li>Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptionally+active+DNA-binding+site+for+human+p53+protein+complexes.">A transcriptionally active DNA-binding site for human p53 protein complexes.</a> Mol. Cell Biol. 12, 2866-2871 (1992).</li><li>DasGupta, R., Kaykas, A., Moon, R. 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Genet. 19, 379-383 (1998).</li><li>Korinek, 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=Two+members+of+the+Tcf+family+implicated+in+Wnt/beta-catenin+signaling+during+embryogenesis+in+the+mouse.">Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse.</a> Mol. Cell Biol. 18, 1248-1256 (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+characterization+of+human+colon+and+rectal+cancer.">Comprehensive molecular characterization of human colon and rectal cancer.</a> Nature. 487, 330-337 (2012).</li><li>Koboldt, D. 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O.K. and L.L are authors on a pending patent relating to multiplexed luciferase technology.

There are several purveyors of luciferase-based reporter systems (such as Promega, Targeting Systems, New England BIolabs, and Thermo Fisher) that provide useful on-line resources for those entertaining a high-throughput screen for the first time. In addition, a number of excellent reviews regarding optimizing the use of high-throughput screens for gene discovery and how RNAi-based screening results can be integrated with other -omics-based datasets should be helpful15,16. Methods for the selection of "hits" f...

A variety of luciferase-based reporters for monitoring a diverse array of cell biological processes are commercially available. The majority of these DNA constructs encode luciferase proteins that are induced to express by specific cell stimuli or perturbations. The most robust transcriptionally based reporters place the expression of a luciferase enzyme under the control of synthetic enhancer elements or gene promoter regions well-established for their reliability in reporting a physiologically relevant transcriptional event1,2. There are also trans-reporter luciferase systems that utilize GAL4-UAS to drive luciferase protein expression. Gal4 is a yeast transcriptional activator and the UAS is an enhancer to which Gal4 specifically binds to activate the transcription of gene sequences placed down-stream of it3. These types of luciferase systems are typically supported by two DNA plasmids - one encodes the GAL4 protein fused to the regulatory portion of a protein of interest, and the second encodes a luciferase protein placed under the control of one or more UAS sequences. Thus, the cellular luciferase signal reflects the activity level of the protein of interest. Another common use of luciferase-based reporters is for monitoring protein stability whereby a protein of interest is fused to a luciferase enzyme4. Regardless of the type of reporter, a caveat emptor is noted here as one cannot assume the specificity of any reporter to have been well interrogated. Thus, due diligence is required in the incorporation of new reporter constructs in any research strategy.
The selection of the luciferase enzyme read-out can be as important as the reliability of the DNA elements that control its expression. Whereas firefly luciferase (FL) is the most commonly used enzyme in commercially available constructs, the advent of new luciferase reporter systems that do not require ATP (as in the case of FL-based reactions) or that incorporate more stable enzymes that emit stronger signals promise to improve the efficiency and reliability of this research platform. An important note regarding the selection of luciferase reporters in chemical studies is the susceptibility of FL to chemical inhibition that could give rise to false reports. In our experience, other enzymes such as Renilla or Gaussia luciferase (RL and GL, respectively) that utilize coelenterazine as a substrate are less readily inhibited by small molecules5.
The most common format for multiplexing luciferase read-outs entails the use of FL and RL largely given the availability of easy-to-use kits with the ability to sequentially measure enzymatic activities from a single sample. In most kits, samples are first exposed to luciferin to reveal levels of FL activity followed by a quenching reagent that is simultaneously deposited with coelenterazine to yield a secondary RL signal. With the addition of other luciferases that can be secreted such as GL or that use yet another substrate such as Cypridina luciferase (CL), a greater number of possibilities for generating high content data using luciferases have emerged. An example of a genome-wide RNAi screen using this technique can be found here6. These technological advances have in turn spurred the development of specific mechanisms to quench each type of luciferase enzyme in order to limit cross-talk7. In our hands, we note a greater frequency of "edge effects" (inexplicable trends in cellular activity associated with the edge of high titer culture plates), with secreted luciferases such as GL or CL. On the other hand, such secreted luciferases are useful for kinetic assays as they enable signal sampling without adulterating cell viability.
For our study presented here, we will simultaneously monitor the activity of three cancer-relevant cellular processes: the p53, Kras, and Wnt signal transduction pathways. The reporters incorporated in our study are the pp53-TA-Luc FL reporter from Clontech (henceforth referred to as the p53-FL reporter)8, an Elk1-GAL4/UAS-CL reporter system (henceforth Elk-1 reporter; Agilent), and the 8X TCF RL reporter that incorporates multiple synthetic enhancer elements recognized by the Wnt pathway transcriptional regulators known as T-cell factors (or TCFs)9-11.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>PathDetect Elk1 Trans-Reporting System</td><td>Agilent Technologies Inc.</td><td>219005</td><td></td></tr><tr><td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td><td>Clontech Lab Inc.</td><td>631914</td><td></td></tr><tr><td>Effectene Transfection Reagent</td><td>Qiagen Inc.</td><td>301427</td><td></td></tr><tr><td>Dual-Luciferase Reporter Assay System</td><td>Promega Corp.</td><td>E1960</td><td></td></tr><tr><td>Cypridina Luciferase Assay reagent</td><td>Targeting Systems</td><td>VLAR-1</td><td></td></tr><tr><td>HCT116 cells</td><td>ATCC</td><td>CCL-247</td><td></td></tr><tr><td>CytoTox-Fluo Cytotoxicity Assay</td><td>Promega Corp.</td><td>G9260</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>MultiFlo Microplate Dispenser</td><td>Labsystems Inc.</td><td>Model 832</td><td></td></tr><tr><td>Biomek FXP Laboratory Automation Workstation</td><td>Beckman Coulter Inc.</td><td>A31842</td><td></td></tr><tr><td>PHERAstar FS-multi-mode HTS microplate reader</td><td>BMG Labtech Inc.</td><td>0471-101A</td><td></td></tr><tr><td>Bright-Line Reichert Hemacytometers</td><td>Hausser Scientific Company</td><td>1490</td><td></td></tr></tbody></table>

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.

Despite advances in mapping the mutational landscape of various cancers using massive genome sequencing efforts12-14, we still do not have in place a systematic approach for translating these observations into intervention strategies. In colorectal cancer (CRC), mutations that are predicted to affect components of three cellular processes - Wnt/&beta;-catenin, p53, and Kras signaling - are found in 99% of all tumors suggesting they are key drivers of cellular transformation in the gut13. Thus, cance...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

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

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  ...

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11.0K Views.  UT Southwestern Medical Center. 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.

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

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.

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.

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

Cancer Research

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 ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Immunology and Infection

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Biology

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Cellular Bioluminescence Based Assay: A High Throughput Method for Combinatorial Drug Screening

Source: Hu, Z. et al. <a href="https://www.jove.com/v/62312/a-rapid-screening-workflow-to-identify-potential-combination-therapy">A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.</a>&#160;J. Vis. Exp.&#160;(2021)
In this video, we describe a method highlighting the utility of bioluminescence assay for sensitive and rapid screening of drugs. These drugs can be tested alone or in combination to assess the synergism in their mode of action.

Source: Hu, Z. et al. A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vitro studies

ATP-Based Luciferase Viability Assay: A Homogenous Method to Evaluate the Growth-Inhibitory Potential of Test Agents on Tumor Organoids

Source: Higa, A. et al. <a href="https://app.jove.com/v/62668">High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids.</a> J. Vis. Exp. (2021)
In this video, we demonstrate a rapid, single-addition assay to assess cell viability in patient-derived organoids upon treatment with a test agent using intracellular ATP levels as a marker for cellular metabolic activity. The intracellular ATP levels are measured using the luciferase/luciferin reaction, where the emitted light intensity indicates the cellular viability and, in turn, the test agent&#39;s growth-inhibitory potency.

Source: Higa, A. et al. High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids. J. Vis. Exp. (2021)
In this video, we demonstrate a rapid, ...