Rapid emergence and mechanisms of resistance by U87 glioblastoma cells to doxorubicin in an in vitro tumor microfluidic ecology

In vitro prediction of the probable rapid emergence of resistance to a drug in tumors could act to winnow out potential candidates for further costly development. We have developed a microfluidic device consisting of ∼500 hexagonal microcompartments that provides a complex ecology with wide ranges o...

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Published in:Proceedings of the National Academy of Sciences - PNAS 2016-12, Vol.113 (50), p.14283-14288
Main Authors: Han, Jeonghun, Jun, Yukyung, Kim, So Hyun, Hoang, Hong-Hoa, Jung, Yeonjoo, Kim, Suyeon, Kim, Jaesang, Austin, Robert H., Lee, Sanghyuk, Park, Sungsu
Format: Article
Language:English
Subjects:
Aldo-Keto Reductases - genetics
Aldo-Keto Reductases - metabolism
Antibiotics, Antineoplastic - pharmacokinetics
Antibiotics, Antineoplastic - pharmacology
Biological Sciences
Brain Neoplasms - drug therapy
Brain Neoplasms - genetics
Brain Neoplasms - metabolism
Cell Line, Tumor
Cells
Chemotherapy
Directed Molecular Evolution
Doxorubicin - pharmacokinetics
Doxorubicin - pharmacology
Drug resistance
Drug Resistance, Neoplasm - genetics
Filamins - genetics
Filamins - metabolism
Gene expression
Glioblastoma - drug therapy
Glioblastoma - genetics
Glioblastoma - metabolism
Humans
Lab-On-A-Chip Devices
Microfluidic Analytical Techniques - methods
Mutation
NF-kappa B - metabolism
Physical Sciences
Signal Transduction
Tumors
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Summary:In vitro prediction of the probable rapid emergence of resistance to a drug in tumors could act to winnow out potential candidates for further costly development. We have developed a microfluidic device consisting of ∼500 hexagonal microcompartments that provides a complex ecology with wide ranges of drug and nutrient gradients and local populations. This ecology of a fragmented metapopulation induced the drug resistance in stage IV U87 glioblastoma cells to doxorubicin in 7 d. Exome and transcriptome sequencing of the resistant cells identified mutations and differentially expressed genes. Gene ontology and pathway analyses of the genes identified showed that they were functionally relevant to the established mechanisms of doxorubicin action. Specifically, we identified (i) a frame-shift insertion in the filamin-A gene, which regulates the influx and efflux of topoisomerase II poisons; (ii) the overexpression of aldo-keto reductase enzymes, which convert doxorubicin into doxorubicinol; and (iii) activation of NF-κB via alterations in the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway from mutations in three genes (CARD6, NSD1, and NLRP13) and the overexpression of inflammatory cytokines. Functional experiments support the in silico analyses and, together, demonstrate the effects of these genetic changes. Our findings suggest that, given the rapid evolution of resistance and the focused response, this technology could act as a rapid screening modality for genetic aberrations leading to resistance to chemotherapy as well as counter selection of drugs unlikely to be successful ultimately.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1614898113