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High-throughput screening using multicellular tumor spheroids to reveal and exploit tumor-specific vulnerabilities
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS.

In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines.

In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , 50 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1334
Keyword [en]
spheroids, high-throughput screening, nitazoxanide, OXPHOS, gene expression profiling, statins, drug repositioning, 3D cell culture, quiescent cells
National Category
Cell and Molecular Biology Medicinal Chemistry Pharmaceutical Sciences Cancer and Oncology Biomedical Laboratory Science/Technology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology Cell Biology
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-320598ISBN: 978-91-554-9921-1 (print)OAI: oai:DiVA.org:uu-320598DiVA: diva2:1090154
Public defence
2017-06-10, Rosénsalen, Ing 95/96, Akademiska Barnsjukhuset, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2017-05-18 Created: 2017-04-22 Last updated: 2017-05-18
List of papers
1. Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer
Open this publication in new window or tab >>Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer
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2015 (English)In: Molecular Cancer Therapeutics, ISSN 1535-7163, E-ISSN 1538-8514, Vol. 14, no 6, 1504-1516 p.Article in journal (Refereed) Published
Abstract [en]

Because dormant cancer cells in hypoxic and nutrient-deprived regions of solid tumors provide a major obstacle to treatment, compounds targeting those cells might have clinical benefits. Here, we describe a high-throughput drug screening approach, using glucose-deprived multicellular tumor spheroids (MCTS) with inner hypoxia, to identify compounds that specifically target this cell population. We used a concept of drug repositioning-using known molecules for new indications. This is a promising strategy to identify molecules for rapid clinical advancement. By screening 1,600 compounds with documented clinical history, we aimed to identify candidates with unforeseen potential for repositioning as anticancer drugs. Our screen identified five molecules with pronounced MCTS-selective activity: nitazoxanide, niclosamide, closantel, pyrvinium pamoate, and salinomycin. Herein, we show that all five compounds inhibit mitochondrial respiration. This suggests that cancer cells in low glucose concentrations depend on oxidative phosphorylation rather than solely glycolysis. Importantly, continuous exposure to the compounds was required to achieve effective treatment. Nitazoxanide, an FDA-approved antiprotozoal drug with excellent pharmacokinetic and safety profile, is the only molecule among the screening hits that reaches high plasma concentrations persisting for up to a few hours after single oral dose. Nitazoxanide activated the AMPK pathway and downregulated c-Myc, mTOR, and Wnt signaling at clinically achievable concentrations. Nitazoxanide combined with the cytotoxic drug irinotecan showed anticancer activity in vivo. We here report that the FDA-approved anthelmintic drug nitazoxanide could be a potential candidate for advancement into cancer clinical trials.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-260735 (URN)10.1158/1535-7163.MCT-14-0792 (DOI)000358054300025 ()25911689 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research CouncilSwedish Childhood Cancer Foundation
Available from: 2015-08-24 Created: 2015-08-24 Last updated: 2017-04-22Bibliographically approved
2. Large-Scale Gene Expression Profiling Platform for Identification of Context-Dependent Drug Responses in Multicellular Tumor Spheroids
Open this publication in new window or tab >>Large-Scale Gene Expression Profiling Platform for Identification of Context-Dependent Drug Responses in Multicellular Tumor Spheroids
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2016 (English)In: CELL CHEMICAL BIOLOGY, ISSN 2451-9448, Vol. 23, no 11, 1428-1438 p.Article in journal (Refereed) Published
Abstract [en]

Cancer cell lines grown as two-dimensional (2D) cultures have been an essential model for studying cancer biology and anticancer drug discovery. However, 2D cancer cell cultures have major limitations, as they do not closely mimic the heterogeneity and tissue context of in vivo tumors. Developing three-dimensional (3D) cell cultures, such as multicellular tumor spheroids, has the potential to address some of these limitations. Here, we combined a high-throughput gene expression profiling method with a tumor spheroid-based drug-screening assay to identify context-dependent treatment responses. As a proof of concept, we examined drug responses of quiescent cancer cells to oxidative phosphorylation (OXPHOS) inhibitors. Use of multicellular tumor spheroids led to discovery that the mevalonate pathway is upregulated in quiescent cells during OXPHOS inhibition, and that OXPHOS inhibitors and mevalonate pathway inhibitors were synergistically toxic to quiescent spheroids. This work illustrates how 3D cellular models yield functional and mechanistic insights not accessible via 2D cultures.

National Category
Cell and Molecular Biology Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-311191 (URN)10.1016/j.chembiol.2016.09.013 (DOI)000388373200015 ()27984028 (PubMedID)
Funder
Swedish Cancer SocietySwedish Foundation for Strategic Research
Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2017-04-22Bibliographically approved
3. Drug combination screening in multicellular tumor spheroids identifies synthetic lethalities in quiescent cancer cells
Open this publication in new window or tab >>Drug combination screening in multicellular tumor spheroids identifies synthetic lethalities in quiescent cancer cells
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(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology Medicinal Chemistry
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-320596 (URN)
Available from: 2017-04-22 Created: 2017-04-22 Last updated: 2017-04-22
4. Targeting tumor cells based on PDE3A expression
Open this publication in new window or tab >>Targeting tumor cells based on PDE3A expression
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(English)Manuscript (preprint) (Other academic)
National Category
Medicinal Chemistry Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-320597 (URN)
Available from: 2017-04-22 Created: 2017-04-22 Last updated: 2017-04-22

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