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Article ; Online: WITHDRAWN: Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines.

Close, David A / Johnston, Paul A

SLAS discovery : advancing life sciences R & D

2023 Volume 29, Issue 1F, Page(s) 130

Language	English
Publishing date	2023-12-13
Publishing country	United States
Document type	Retraction of Publication
ZDB-ID	2885123-7
ISSN	2472-5560 ; 2472-5552
ISSN (online)	2472-5560
ISSN	2472-5552
DOI	10.1016/j.slasd.2023.12.002
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Article ; Online: Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines.

Close, David A / Johnston, Paul A

SLAS discovery : advancing life sciences R & D

2021 Volume 27, Issue 1, Page(s) 39–54

Abstract: In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or ...

Abstract	In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for cancer drug discovery experience 20%-21% O
MeSH term(s)	Antineoplastic Agents/pharmacology ; Antineoplastic Agents/therapeutic use ; Head and Neck Neoplasms/drug therapy ; Head and Neck Neoplasms/genetics ; Humans ; Hypoxia/drug therapy ; Spheroids, Cellular ; Squamous Cell Carcinoma of Head and Neck/drug therapy ; Squamous Cell Carcinoma of Head and Neck/genetics ; Tumor Microenvironment
Chemical Substances	Antineoplastic Agents
Language	English
Publishing date	2021-10-13
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2885123-7
ISSN	2472-5560 ; 2472-5552
ISSN (online)	2472-5560
ISSN	2472-5552
DOI	10.1016/j.slasd.2021.10.008
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Article ; Online: Maximizing the Value of Cancer Drug Screening in Multicellular Tumor Spheroid Cultures: A Case Study in Five Head and Neck Squamous Cell Carcinoma Cell Lines.

Kochanek, Stanton J / Close, David A / Camarco, Daniel P / Johnston, Paul A

SLAS discovery : advancing life sciences R & D

2020 Volume 25, Issue 4, Page(s) 329–349

Abstract: With approval rates <5% and the probability of success in oncology clinical trials of 3.4%, more physiologically relevant in vitro three-dimensional models are being deployed during lead generation to select better drug candidates for solid tumors. ... ...

Abstract	With approval rates <5% and the probability of success in oncology clinical trials of 3.4%, more physiologically relevant in vitro three-dimensional models are being deployed during lead generation to select better drug candidates for solid tumors. Multicellular tumor spheroids (MCTSs) resemble avascular tumor nodules, micrometastases, or the intervascular regions of large solid tumors with respect to morphology, cell-cell and cell-extracellular matrix contacts, and volume growth kinetics. MCTSs develop gradients of nutrient and oxygen concentration resulting in diverse microenvironments with differential proliferation and drug distribution zones. We produced head and neck squamous cell carcinoma (HNSCC) MCTSs in 384-well U-bottom ultra-low-attachment microtiter plates and used metabolic viability and imaging methods to measure morphologies, growth phenotypes and the effects of 19 anticancer drugs. We showed that cell viability measurements underestimated the impact of drug exposure in HNSCC MCTS cultures, but that incorporating morphology and dead-cell staining analyses increased the number of drugs judged to have substantially impacted MCTS cultures. A cumulative multiparameter drug impact score enabled us to stratify MCTS drug responses into high-, intermediate-, and low-impact tiers, and maximized the value of these more physiologically relevant tumor cultures. It is conceivable that the viable cells present in MCTS cultures after drug exposure arise from drug-resistant populations that could represent a source of drug failure and recurrence. Long-term monitoring of treated MCTS cultures could provide a strategy to determine whether these drug-resistant populations represent circumstances where tumor growth is delayed and may ultimately give rise to regrowth.
MeSH term(s)	Antineoplastic Agents/pharmacology ; Apoptosis/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Drug Screening Assays, Antitumor ; Early Detection of Cancer ; Humans ; Spheroids, Cellular/drug effects ; Squamous Cell Carcinoma of Head and Neck/drug therapy ; Squamous Cell Carcinoma of Head and Neck/pathology ; Tumor Microenvironment/drug effects
Chemical Substances	Antineoplastic Agents
Language	English
Publishing date	2020-01-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2885123-7
ISSN	2472-5560 ; 2472-5552
ISSN (online)	2472-5560
ISSN	2472-5552
DOI	10.1177/2472555219896999
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Article ; Online: High Content Screening Characterization of Head and Neck Squamous Cell Carcinoma Multicellular Tumor Spheroid Cultures Generated in 384-Well Ultra-Low Attachment Plates to Screen for Better Cancer Drug Leads.

Kochanek, Stanton J / Close, David A / Johnston, Paul A

Assay and drug development technologies

2018 Volume 17, Issue 1, Page(s) 17–36

Abstract: Multicellular tumor spheroid (MCTS) cultures represent more physiologically relevant in vitro cell tumor models that recapitulate the microenvironments and cell-cell or cell-extracellular matrix interactions which occur in solid tumors. We characterized ... ...

Abstract	Multicellular tumor spheroid (MCTS) cultures represent more physiologically relevant in vitro cell tumor models that recapitulate the microenvironments and cell-cell or cell-extracellular matrix interactions which occur in solid tumors. We characterized the morphologies, viability, and growth behaviors of MCTSs produced by 11 different head and neck squamous cell carcinoma (HNSCC) cell lines seeded into and cultured in ultra-low attachment microtiter plates (ULA-plates) over extended periods of time. HNSCC MCTS cultures developed microenvironments, which resulted in differences in proliferation rates, metabolic activity, and mitochondrial functional activity between cells located in the outer layers of the MCTS and cells in the interior. HNSCC MCTS cultures exhibited drug penetration and distribution gradients and some developed necrotic cores. Perhaps the most profound effect of culturing HNSCC cell lines in MCTS cultures was their dramatically altered and varied growth phenotypes. Instead of the exponential growth that are characteristic of two-dimensional HNSCC growth inhibition assays, some MCTS cultures displayed linear growth rates, categorized as rapid, moderate, or slow, dormant MCTSs remained viable but did not grow, and some MCTSs exhibited death phenotypes that were either progressive and slow or rapid. The ability of MCTS cultures to develop microenvironments and to display a variety of different growth phenotypes provides in vitro models that are more closely aligned with solid tumors in vivo. We anticipate that the implementation MCTS models to screen for new cancer drugs for solid tumors like HNSCC will produce leads that will translate better in in vivo animal models and patients.
MeSH term(s)	Antibiotics, Antineoplastic/pharmacology ; Cell Culture Techniques ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Doxorubicin/pharmacology ; Drug Screening Assays, Antitumor ; High-Throughput Screening Assays ; Humans ; Spheroids, Cellular/drug effects ; Squamous Cell Carcinoma of Head and Neck/drug therapy ; Squamous Cell Carcinoma of Head and Neck/pathology ; Tumor Cells, Cultured
Chemical Substances	Antibiotics, Antineoplastic ; Doxorubicin (80168379AG)
Language	English
Publishing date	2018-12-28
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1557-8127
ISSN (online)	1557-8127
DOI	10.1089/adt.2018.896
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Article ; Online: Unbiased High-Throughput Drug Combination Pilot Screening Identifies Synergistic Drug Combinations Effective against Patient-Derived and Drug-Resistant Melanoma Cell Lines.

Close, David A / Kirkwood, John M / Fecek, Ronald J / Storkus, Walter J / Johnston, Paul A

SLAS discovery : advancing life sciences R & D

2020 Volume 26, Issue 5, Page(s) 712–729

Abstract: We describe the development, optimization, and validation of 384-well growth inhibition assays for six patient-derived melanoma cell lines (PDMCLs), three wild type (WT) ... ...

Abstract	We describe the development, optimization, and validation of 384-well growth inhibition assays for six patient-derived melanoma cell lines (PDMCLs), three wild type (WT) for
MeSH term(s)	Animals ; Antineoplastic Agents/chemistry ; Antineoplastic Agents/pharmacology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm/drug effects ; Drug Screening Assays, Antitumor/methods ; Drug Screening Assays, Antitumor/standards ; Drug Synergism ; High-Throughput Screening Assays/methods ; High-Throughput Screening Assays/standards ; Humans ; Melanoma/drug therapy ; Mice ; Reproducibility of Results
Chemical Substances	Antineoplastic Agents
Language	English
Publishing date	2020-11-18
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2885123-7
ISSN	2472-5560 ; 2472-5552
ISSN (online)	2472-5560
ISSN	2472-5552
DOI	10.1177/2472555220970917
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Article ; Online: Characterization of allosteric modulators that disrupt androgen receptor co-activator protein-protein interactions to alter transactivation-Drug leads for metastatic castration resistant prostate cancer.

Fancher, Ashley T / Hua, Yun / Close, David A / Xu, Wei / McDermott, Lee A / Strock, Christopher J / Santiago, Ulises / Camacho, Carlos J / Johnston, Paul A

SLAS discovery : advancing life sciences R & D

2023

Abstract: Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional ... ...

Abstract	Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4-2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H
Language	English
Publishing date	2023-08-06
Publishing country	United States
Document type	Journal Article
ZDB-ID	2885123-7
ISSN	2472-5560 ; 2472-5552
ISSN (online)	2472-5560
ISSN	2472-5552
DOI	10.1016/j.slasd.2023.08.001
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Article ; Online: High-Content Screening Comparison of Cancer Drug Accumulation and Distribution in Two-Dimensional and Three-Dimensional Culture Models of Head and Neck Cancer.

Shan, Feng / Close, David A / Camarco, Daniel P / Johnston, Paul A

Assay and drug development technologies

2017 Volume 16, Issue 1, Page(s) 27–50

Abstract: High cancer drug development attrition rates have provoked considerable debate about whether the two-dimensional tumor growth inhibition high-throughput screening assays used in pre-clinical lead discovery adequately reflect solid tumor complexity. We ... ...

Abstract	High cancer drug development attrition rates have provoked considerable debate about whether the two-dimensional tumor growth inhibition high-throughput screening assays used in pre-clinical lead discovery adequately reflect solid tumor complexity. We used automated high-content screening image acquisition and analysis methods to compare fluorescent drug uptake, accumulation, and distribution in Cal33 and FaDu head and neck cancer (HNC) monolayer and multicellular tumor spheroid (MCTS) models. Ellipticine, idarubicin, daunorubicin, and doxorubicin were studied because of their fluorescent properties and broad anti-tumor activities. HNC MCTSs were generated in 384-well ultra-low attachment plates where compound exposure, image acquisition, and analysis could be performed in situ. Fluorescent drug accumulation in Cal33 monolayer and MCTS cultures was linear with respect to concentration, and appeared to achieve steady-state levels within 10-15 min of drug exposure, which were maintained through 30-45 min. Drug accumulation in monolayers was independent of cell number and/or density, and every cell achieved uniform drug concentrations. In MCTSs, however, drug accumulation increased as the number of cells and sizes of the MCTSs became bigger. Drugs exhibited restricted penetration and distribution gradients, accumulating preferentially in cells in the outer layers of MCTSs relative to those in the inner cores. Cal33 monolayers were 6-, 20-, 10-, and 16-fold more sensitive than MCTSs to growth inhibition by ellipticine, idarubicin, daunorubicin, and doxorubicin, respectively. In Cal33 MCTSs exposed to ellipticine or doxorubicin for 24 h, MCTSs were smaller and although they still exhibited drug penetration and distribution gradients, the fluorescent intensity difference between outer and inner cells was reduced. After a 24 h exposure, both drugs had penetrated throughout FaDu MCTSs, consistent with drug-induced death of peripheral cell layers enhancing drug penetration. The increased resistance of MCTS cultures and their ability to recapitulate drug penetration and distribution gradients argues strongly for the deployment of these more physiological models in cancer lead discovery. MCTSs have the potential to enhance the correlation between in vitro potencies and in vivo efficacy, and ultimately may lead to improved cancer drug approval rates.
MeSH term(s)	Antineoplastic Agents/pharmacokinetics ; Cell Culture Techniques ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Dose-Response Relationship, Drug ; Drug Discovery ; Drug Screening Assays, Antitumor ; Head and Neck Neoplasms/metabolism ; Head and Neck Neoplasms/pathology ; High-Throughput Screening Assays ; Humans ; Models, Biological ; Structure-Activity Relationship ; Tissue Distribution
Chemical Substances	Antineoplastic Agents
Language	English
Publishing date	2017-12-07
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural
ISSN	1557-8127
ISSN (online)	1557-8127
DOI	10.1089/adt.2017.812
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Article ; Online: Selecting a minimal set of androgen receptor assays for screening chemicals.

Judson, Richard / Houck, Keith / Paul Friedman, Katie / Brown, Jason / Browne, Patience / Johnston, Paul A / Close, David A / Mansouri, Kamel / Kleinstreuer, Nicole

Regulatory toxicology and pharmacology : RTP

2020 Volume 117, Page(s) 104764

Abstract: Screening certain environmental chemicals for their ability to interact with endocrine targets, including the androgen receptor (AR), is an important global concern. We previously developed a model using a battery of eleven in vitro AR assays to predict ... ...

Abstract	Screening certain environmental chemicals for their ability to interact with endocrine targets, including the androgen receptor (AR), is an important global concern. We previously developed a model using a battery of eleven in vitro AR assays to predict in vivo AR activity. Here we describe a revised mathematical modeling approach that also incorporates data from newly available assays and demonstrate that subsets of assays can provide close to the same level of predictivity. These subset models are evaluated against the full model using 1820 chemicals, as well as in vitro and in vivo reference chemicals from the literature. Agonist batteries of as few as six assays and antagonist batteries of as few as five assays can yield balanced accuracies of 95% or better relative to the full model. Balanced accuracy for predicting reference chemicals is 100%. An approach is outlined for researchers to develop their own subset batteries to accurately detect AR activity using assays that map to the pathway of key molecular and cellular events involved in chemical-mediated AR activation and transcriptional activity. This work indicates in vitro bioactivity and in silico predictions that map to the AR pathway could be used in an integrated approach to testing and assessment for identifying chemicals that interact directly with the mammalian AR.
MeSH term(s)	Androgen Receptor Antagonists/metabolism ; Androgen Receptor Antagonists/toxicity ; Androgens/metabolism ; Androgens/toxicity ; Animals ; Environmental Exposure/prevention & control ; Environmental Exposure/statistics & numerical data ; Hazardous Substances/metabolism ; Hazardous Substances/toxicity ; High-Throughput Screening Assays/methods ; Humans ; Models, Theoretical ; Receptors, Androgen/metabolism
Chemical Substances	Androgen Receptor Antagonists ; Androgens ; Hazardous Substances ; Receptors, Androgen
Language	English
Publishing date	2020-08-14
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	604672-1
ISSN	1096-0295 ; 0273-2300
ISSN (online)	1096-0295
ISSN	0273-2300
DOI	10.1016/j.yrtph.2020.104764
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Article ; Online: Confirmation of Selected Synergistic Cancer Drug Combinations Identified in an HTS Campaign and Exploration of Drug Efflux Transporter Contributions to the Mode of Synergy.

Kochanek, Stanton J / Close, David A / Wang, Allen Xinwei / Shun, Tongying / Empey, Philip E / Eiseman, Julie L / Johnston, Paul A

SLAS discovery : advancing life sciences R & D

2019 Volume 24, Issue 6, Page(s) 653–668

Abstract: Systematic unbiased high-throughput screening (HTS) of drug combinations (DCs) in well-characterized tumor cell lines is a data-driven strategy to identify novel DCs with potential to be developed into effective therapies. Four DCs from a DC HTS campaign ...

Abstract	Systematic unbiased high-throughput screening (HTS) of drug combinations (DCs) in well-characterized tumor cell lines is a data-driven strategy to identify novel DCs with potential to be developed into effective therapies. Four DCs from a DC HTS campaign were selected for confirmation; only one appears in clinicaltrials.gov and limited preclinical in vitro data indicates that the drug pairs interact synergistically. Nineteen DC-tumor cell line sets were confirmed to interact synergistically in three pharmacological interaction models. We developed an imaging assay to quantify accumulation of the ABCG2 efflux transporter substrate Hoechst. Gefitinib and raloxifene enhanced Hoechst accumulation in ABCG2 (BCRP)-expressing cells, consistent with inhibition of ABCG2 efflux. Both drugs also inhibit ABCB1 efflux. Mitoxantrone, daunorubicin, and vinorelbine are substrates of one or more of the ABCG2, ABCB1, or ABCC1 efflux transporters expressed to varying extents in the selected cell lines. Interactions between ABC drug efflux transporter inhibitors and substrates may have contributed to the observed synergy; however, other mechanisms may be involved. Novel synergistic DCs identified by HTS were confirmed in vitro, and plausible mechanisms of action studied. Similar approaches may justify the testing of novel HTS-derived DCs in mouse xenograft human cancer models and support the clinical evaluation of effective in vivo DCs in patients.
MeSH term(s)	ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics ; ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism ; ATP-Binding Cassette Transporters/genetics ; ATP-Binding Cassette Transporters/metabolism ; Animals ; Antineoplastic Agents/pharmacology ; Cell Culture Techniques ; Cell Line, Tumor ; Drug Resistance, Multiple/genetics ; Drug Resistance, Neoplasm/genetics ; Drug Screening Assays, Antitumor/methods ; Drug Synergism ; High-Throughput Screening Assays ; Humans ; Molecular Imaging ; Pilot Projects
Chemical Substances	ATP Binding Cassette Transporter, Subfamily G, Member 2 ; ATP-Binding Cassette Transporters ; Antineoplastic Agents
Language	English
Publishing date	2019-04-30
Publishing country	United States
Document type	Journal Article ; Research Support, American Recovery and Reinvestment Act ; Research Support, N.I.H., Extramural
ZDB-ID	2885123-7
ISSN	2472-5560 ; 2472-5552
ISSN (online)	2472-5560
ISSN	2472-5552
DOI	10.1177/2472555219844566
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Article ; Online: The Generation of Three-Dimensional Head and Neck Cancer Models for Drug Discovery in 384-Well Ultra-Low Attachment Microplates.

Close, David A / Camarco, Daniel P / Shan, Feng / Kochanek, Stanton J / Johnston, Paul A

Methods in molecular biology (Clifton, N.J.)

2017 Volume 1683, Page(s) 355–369

Abstract: The poor success rate of cancer drug discovery has prompted efforts to develop more physiologically relevant cellular models for early preclinical cancer lead discovery assays. For solid tumors, this would dictate the implementation of three-dimensional ( ...

Abstract	The poor success rate of cancer drug discovery has prompted efforts to develop more physiologically relevant cellular models for early preclinical cancer lead discovery assays. For solid tumors, this would dictate the implementation of three-dimensional (3D) tumor models that more accurately recapitulate human solid tumor architecture and biology. A number of anchorage-dependent and anchorage-independent in vitro 3D cancer models have been developed together with homogeneous assay methods and high content imaging approaches to assess tumor spheroid morphology, growth, and viability. However, several significant technical challenges have restricted the implementation of some 3D models in HTS. We describe a method that uses 384-well U-bottomed ultra-low attachment (ULA) microplates to produce head and neck tumor spheroids for cancer drug discovery assays. The production of multicellular head and neck cancer spheroids in 384-well ULA-plates occurs in situ, does not impose an inordinate tissue culture burden for HTS, is readily compatible with automation and homogeneous assay detection methods, and produces high-quality uniform-sized spheroids that can be utilized in cancer drug cytotoxicity assays within days rather than weeks.
MeSH term(s)	Antineoplastic Agents/pharmacology ; Cell Culture Techniques ; Cell Line, Tumor ; Cell Survival/drug effects ; Drug Discovery/methods ; Drug Screening Assays, Antitumor/methods ; Head and Neck Neoplasms ; High-Throughput Screening Assays ; Humans ; Microscopy ; Spheroids, Cellular/drug effects
Chemical Substances	Antineoplastic Agents
Language	English
Publishing date	2017-10-30
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-7357-6_20
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