Dan Lazar

A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells ☆

Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1-/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators.

Abstract 3731: Luminescent cell health assays for tumor spheroid evaluation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Microtissues produced in 3D cell culture are much more representative of actual living tissue compared to monolayers produced in 2D cell culture. In fact, in the area of oncology research, multicellular tumor spheroids are considered an excellent platform for testing drug delivery and efficacy. As the necessity for established 3D cell culture models rises, there is also a need for convenient assays that have been specifically demonstrated to be effective for use with 3D microtissues. The more complex architecture of 3D microtissues demands increased lytic effectiveness and reagent penetration, characteristics that are often only minor considerations for reagents designed for 2D cell culture. Here we report on a variety of bioluminescent and fluorescent cell-based assays applied to hanging-drop spheroids produced from HCT116 colon cancer cells. The first assay to be described is an ATP detection reagent for measuring cell viability. This reagent has both an improved formulation and an optimized assay protocol and has clear advantages over other viability assays. Other cell health assays will also be described, including reagents that measure cell death, apoptosis, mechanistic cytotoxicity, or reporter gene expression. These additional assays do not require a change in formulation, but do require new protocols in order to optimize their effectiveness when applied to 3D microtissues. As with their application to cells in 2D culture, these “add-mix-measure” reagents are robust and amenable to both low- and high-throughput applications. Citation Format: Michael P. Valley, Kevin R. Kupcho, Chad A. Zimprich, Andrew L. Niles, James J. Cali, Jens M. Kelm, Wolfgang Moritz, Dan F. Lazar. Luminescent cell health assays for tumor spheroid evaluation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3731. doi:10.1158/1538-7445.AM2014-3731

Abstract 4784: Selective bioluminogenic HDAC activity assays for profiling HDAC inhibitors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Histone deacetylases (HDACs) play critical roles in the regulation of gene transcription and cell signaling events by deacetylating histones and other important non-histone substrates. Aberrant increases in HDAC enzyme activities are therefore implicated in a number of human infirmities, including cancers, metabolic disease and neurodegeneration. Fortunately, HDAC enzymes represent attractive pharmacological targets because they are readily tractable with small molecule inhibitors. In fact, several HDAC inhibitors (HDACi) have recently proceeded through (or are near) the FDA approval process for the treatment of hematologic malignancies. However, the promise of clinical HDACi therapy has been hampered by significant dose-limiting toxicities. These off-target effects have led to a renewed focus on basic HDAC biology and the development of isoenzyme-specific HDAC inhibitors which could avoid off-target effects. To help facilitate the discovery of compounds with better defined selectivity profiles, we have developed lysine deacetylase assays that selectively measure specific isoenzyme activities in cells, extracts, or purified recombinant preparations. These assays are based on substrates that are selective due to a combination of extended peptide sequence and novel chemical modifications. Deacetylase activity is measured by delivering a single, pro-luminogenic, homogeneous assay reagent to assay wells, resulting in luminescence proportional to HDAC activity. In addition to being isoenzyme selective, these novel substrates are cell permeable allowing for lytic and non-lytic cell-based HDAC assays. Lastly, these assays are also fully compatible with fluorescent viability and/or cytotoxicity assays. This provides additional flexibility for multiplexed formats which examine not only selective HDAC inhibition, but the functional consequences they exert on cell health. Citation Format: Kevin R. Kupcho, Nathan J. Evans, Andrew L. Niles, Thomas A. Kirkland, Dan F. Lazar. Selective bioluminogenic HDAC activity assays for profiling HDAC inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4784. doi:10.1158/1538-7445.AM2014-4784

Abstract 5532: A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity.

Cell-based assay models are now universally employed as a routine means of establishing and ranking on- and off-target toxicities. It is well appreciated, however, that individual cytotoxic phenotypes are shaped not only by compound dosage, but by the length of compound-cell exposure. In addition to compound-specific attributes, inherent cellular factors such as biotransformation capacity, cell cycle susceptibility, and receptor expression often dictate the kinetics of cytotoxicity. Unfortunately, the majority of conventional cytotoxicity assays are performed at a terminal endpoint (48-72h) which at worst can underestimate cytotoxicity due to biomarker degradation, or at best, fail to reveal important features of the cytotoxic event which may be important for establishing mechanism-of-action. We have developed a pro-fluorescent, cell-impermeant probe which can be applied to cells at the time of dosing to report changes in membrane integrity as they occur in real-time. The dye enters cells with impaired membrane integrity, greatly enhancing fluorescence which is proportional to non-viable cell number. The dye can be used in traditional plate based formats using standard fluorometry or with image based readers (Essen Bioscience Incucyte™) to establish the dose-dependent kinetics of cytotoxicity. Here we describe the dye9s utility using mechanistically distinct cytotoxins with both cancer cell lines (on-target) and terminally differentiated cells (off-target) and correlate this dead cell fluorescence activity to other cytotoxicity biomarkers by same-well multiplexed methods. Citation Format: Richard L. Somberg, Andrew Niles, Tracy Worzella, Dan Lazar. A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5532. doi:10.1158/1538-7445.AM2013-5532

Abstract 3312: A novel plate-based assay for screening autophagic activity in 2D and 3D cell culture models

The critical importance of autophagy in cell health and its proposed role in disease-relevant biology, including cancer, inflammation, and immunology, has increased the need for more effective assays to screen for agents that modulate autophagic activity. Here we utilize NanoLuc Binary Technology (NanoBiT) to develop a homogeneous plate-based assay to measure autophagic flux in cell culture models. In this approach, an exogenous LC3B (Atg8) fusion protein was tagged on its N-terminus with an 11 amino acid peptide (HiBiT) and stably expressed in mammalian cells, including U2OS and HEK293. After exposure to various treatment conditions, cellular levels of this novel autophagy reporter were determined by addition of a lytic detection reagent containing Large BiT (LgBiT). LgBiT rapidly associates with HiBiT in the cell lysate, producing a bright, luminescent enzyme in the presence of the furimazine substrate. The bright signal allows low levels of expression of the reporter, maximizing the assay response, and the signal is stable, allowing assay of multiple 96- or 384-well plates in the same experiment. In response to autophagic stimuli, including nutrient deprivation and various mTORC inhibitors (e.g., PP242 and rapamycin), autophagic degradation of expressed LC3 reporter was evident by reduced assay signal. In contrast, in response to both upstream (e.g., 3-MA and wortmannin) and downstream (e.g., bafilomycin A1 and chloroquine) inhibitors of the autophagy pathway, degradation of the autophagic reporter was effectively blocked and assay signal was consistently increased as predicted. Compound effects were time dependent and stratified according to expected potency and efficacy of the test agents employed. The use of a mutant reporter based on LC3G120A further demonstrated the specificity of the wild-type LC3 reporter for the detection of autophagic activity. When assayed in 384-well plates with automation, HEK293 autophagy reporter cells produced Z’ values of ~0.7 in response to autophagy induction with PP242, while subsequent blockade of autophagy with bafilomycin A1 resulted in Z’ values of ~0.8. This data, and subsequent LOPAC library screening, indicates the potential utility of this assay method for HTS applications. In addition, the HEK293 autophagy reporter cells can be induced to form 3D cell spheroids, thus allowing investigation of assay performance in this more complex model. Autophagy reporter levels increased with increasing spheroid size (up to 650 μm diameter tested) in a manner proportional to a surrogate measure of viable cell number. Importantly, both induction and inhibition of autophagic activity was easily detected following PP242 and bafilomycin A1 treatment, respectively. Using this novel plate-based assay system for the determination of autophagic flux, it is possible to screen test agents and quantitatively determine both the potency and efficacy of autophagy modulation. Citation Format: Dan F. Lazar, Amani A. Gillette, Braeden L. Butler, Christopher T. Eggers, Brock F. Binkowski, Gediminas Vidugiris, Michael R. Slater, Dongping Ma, James J. Cali. A novel plate-based assay for screening autophagic activity in 2D and 3D cell culture models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3312. doi:10.1158/1538-7445.AM2017-3312

Abstract 3505: A bioluminescent, homogeneous annexin V microplate-based method for assessment of apoptosis

The selective elimination of malignant cells via the apoptotic process continues to be the cornerstone of modern anti-cancer therapy regimens. Therefore, in vitro screening approaches aimed at identifying clinically useful apoptosis inducers remain critically important. Recently, phenotypic screening methods have enjoyed a resurgence due to more biologically complex and relevant cell models as well as advances in chemical proteomics which have allowed for more successful target identification. As a consequence, novel probes and tools with enabling attributes are required to fully realize this discovery potential. In an effort to address this unmet need, we have developed a bioluminescent and homogeneous annexin V binding assay for the assessment of apoptosis. Unlike traditional fluorescent annexin V methodology, the “no-wash” reagent employed in this new assay utilizes binary components of a novel luciferase separately fused to annexin V. The annexin V-luciferase subunit fusion pairs have low intrinsic affinity for each other and thus produce no or low luminescence until phosphatidylserine (PtdSer) exposure drives annexin-fusion pair oligimerization. Ultimately, this protein:protein interaction on or near the cell surface reconstitutes full luciferase activity causing an increase in luminescence in the presence of a luciferase substrate. A separate, pro-fluorescent, multiplexed component of the reagent further delineates differences in annexin positivity based on maintenance or loss of membrane integrity corresponding to apoptosis or necrosis, respectively. We validated this method using a panel of diverse cancer cell lines (U2-OS, DLD-1, HeLa, Jurkat, K562, A549, and PC-3), representing both attachment-dependent and -independent morphologies after dose-dependent challenge with intrinsic (bortezomib, panobinostat, staurosporine, and paclitaxel) and extrinsic (rhTRAIL) inducers of apoptosis as well as agents known to produce primary necrosis (ionomycin and digitonin). Caspase activation data was also collected in parallel plates at endpoint as a well-validated and sensitive orthogonal comparator. The bioluminescent annexin V method proved sufficiently robust in 384 well microplate formats to routinely produce Z’ > 0.7 and rank-order potencies in good agreement with caspase activation values. In addition to this microplate functionality, the reagent allowed for sensitive, facile imaging of apoptotic induction in living cells using different imaging platforms. Taken together, the method and reagent should provide unparalleled flexibility with regard to live cell apoptosis detection in both conventional microplate and high content-like imaging formats and advance the pace of new chemical entity discovery. Citation Format: Kevin Kupcho, John Shultz, Andrew Niles, Wenhui Zhou, Robin Hurst, Jim Hartnett, Thomas Machleidt, Terry Riss, Dan Lazar, Jim Cali. A bioluminescent, homogeneous annexin V microplate-based method for assessment of apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3505.

Abstract 1319: Monitoring inflammasome activation with a bioluminescent caspase-1 assay

Inflammatory responses and immune modulation play important and complex roles in cancer development and therapy, but methods to monitor caspase-1 activity associated with inflammasome activation have been limited. Inflammasomes are protein complexes induced by diverse inflammatory stimuli. Caspase-1, an essential component of the inflammasome, is activated in response to these stimuli, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. Western blots and ELISA are the primary, but indirect, methods for monitoring caspase-1 activity currently in use. To simplify and provide a more direct means of detecting cell-based caspase-1 activity, we developed a sensitive, homogeneous, plate-based assay that eliminates the need for significant sample processing. The assay employs a single-step, bioluminescent format combining a caspase-1 substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized reagent subsequently added to treated cells in an assay well. The coupled-enzyme system quickly reaches a steady-state between caspase cleavage of the substrate and luciferase conversion of the aminoluciferin, with light generated proportional to the amount of caspase-1 activity present in the sample. In addition to substrate selection, assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor, MG132, in the reagent and by the subsequent use of a caspase-1 inhibitor, Ac-YVAD-CHO, to confirm activity. This approach enables clear determination of caspase-1 activity even in the context of apoptotic cells. Studies with Casp1 −/− cells further demonstrate the effectiveness of this assay system to specifically detect cell-based, caspase-1 activity. Using this novel assay system, caspase-1 activation has been quantitatively determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, monosodium urate crystals, R-848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages from mice, as well as in human primary monocytes. Of note, caspase-1 activity can be monitored either directly in cells or released into the culture medium following cell treatment with various inflammatory stimuli. Monitoring released caspase-1 activity in supernatants is fast, sensitive, and nondestructive, thereby enabling subsequent multiplexing of the biological sample with other assays to monitor additional cell parameters, such as IL-1β release or cell death. Therefore, this assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators. Citation Format: Martha O9Brien, Danielle Moehring, Raul Munoz-Planillo, Gabriel Nunez, Justin Callaway, Jenny Ting, Mike Scurria, Tim Ugo, Laurent Bernad, James Cali, Dan Lazar. Monitoring inflammasome activation with a bioluminescent caspase-1 assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1319. doi:10.1158/1538-7445.AM2015-1319

Abstract 4237: Isoenzyme-selective HDAC activity assays.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Histone deacetylases (HDACs) are the family of enzymes which catalyze removal of acetyl groups from lysine residues on both histone and non-histone proteins. The lysine acetylation state of histones is an important post-translational modification which affects both chromatin structure and gene transcription. Thus, by deacetylating the amino-terminal tails of histones, HDACs have a profound effect on numerous and diverse cellular functions including cell proliferation. Given this important regulatory role, as well as evidence for significant HDAC involvement in the modulation of cancer, rapid advancement has been made in the development of broad-spectrum HDAC inhibitors. Recently, there has been an increased focus on the development of isoenzyme-specific HDAC inhibitors which may lead to improved therapy, including better efficacy and reduced off target effects. To enable selective HDAC studies, we developed assays that selectively measure specific isoenzyme activities in cells, extracts, or recombinant proteins. The assays use a homogeneous luminescent approach with a single reagent addition that is amenable to HTS. Isoenzyme-selective, luminogenic acetylated peptide substrates provide the basis for HDAC isoenzyme selectivity. Peptide deacetylation is measured by exposure to developer and a luciferin detection reagent. In addition to isoenzyme specificity, the assays show approximately 100-fold or better enhanced sensitivity over fluorescence methods and the versatility necessary for both biochemical and cell-based formats. Citation Format: Kevin R. Kupcho, Nathan J. Evans, Andrew L. Niles, Dan F. Lazar, Thomas A. Kirkland. Isoenzyme-selective HDAC activity assays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4237. doi:10.1158/1538-7445.AM2013-4237

Abstract 5531: A bioluminescent cell viability assay optimized for 3D microtissues.

It has been well demonstrated that the morphology, gene expression, and overall biological response of cells assessed in 3-dimensional (3D) culture models are frequently more physiologically relevant than that of cells studied in standard 2D culture formats. In spite of rapidly growing interest, one hindrance to the use of 3D models for drug discovery and other research efforts is the lack of convenient and effective assays explicitly validated for application to 3D microtissues. Here we report on a bioluminescent ATP-detection assay comprised of both an improved formulation and an optimized protocol specifically designed to measure the viability of cells grown in 3D culture. The ability of the 3D-optimized reagent to effectively lyse cells from throughout the full thickness of 3D microtissues, including cancer cell spheroids, was clearly demonstrated by use of confocal laser fluorescent microscopy in conjunction with a fluorogenic, membrane-impermeant DNA-binding dye. Importantly, the assay9s ability to accurately report the ATP content, and therefore viability, of various 3D microtissues (including a range of cancer cell line spheroids) was demonstrated by comparison to a trichloroacetic acid ATP extraction method. In scaffold-free as well as scaffold-dependent 3D models, recovery and detection of greater than 80% of ATP present was routinely observed. In contrast, another frequently used ATP-detection assay was found to have greatly reduced lytic and ATP detection properties ( Citation Format: Michael P. Valley, Chad Zimprich, James J. Cali, Dan F. Lazar. A bioluminescent cell viability assay optimized for 3D microtissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5531. doi:10.1158/1538-7445.AM2013-5531

Abstract 3885: A bi-functional fluorescent assay for the assessment of cytotoxicity and proliferation

Cell-based models continue to be critical tools for cancer research. Therefore, the assessment of cytotoxic or cytostatic effects remains an obligate experimental activity. Although numerous enzyme activity chemistries exist for measuring cytotoxicity, biomarker degradation may cause underestimation of actual cytotoxicity and limit the utility of these activity measures when ascertained with long-term drug exposures (72hrs or more). We have developed a cell-impermeant probe which labels the DNA from cells with compromised membranes. The quantum efficiency (i.e. brightness) of this dye increases dramatically upon DNA binding, producing a fluorescent signal which can be measured using standard “green” fluorometry wavelengths (excitation 485nm/emission 530nm). Additionally, the dye is non-toxic and stable, allowing for introduction of the probe directly into the culture medium or compound dilutions prior to cell dosing. This “no step” addition feature allows for the measurement of cytotoxicity in real time or at a convenient endpoint. Furthermore, the dye is fully compatible with luminescent measures, allowing for same-well, multi-parametric assessment of the cytotoxic phenotype. Lastly, proliferative or anti-proliferative effects (relative to control) can be revealed by the introduction of a lytic detergent which enables the normally impermeant dye to bind to all DNA in the assay well. Herein, we detail our efforts to characterize and multiplex this dye when applied to suspension and adherent cell lines treated with agents which cause either primary or secondary necrosis in a 72hr time course. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3885. doi:1538-7445.AM2012-3885