期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：50
页码：14283-14288
DOI：10.1073/pnas.1614898113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceDrug resistance is among the most critical problems in cancer treatment. This proof-of-principle study demonstrates that a combination of in vitro microecology and deep sequencing could provide an extremely efficient method to elucidate the in vivo resistance mechanisms of cancer drugs and predict if drug resistance is likely to occur. Remarkably, drug resistance was seen within 7 d in our microfluidic chip, enabling characterization of molecular mechanisms within a month. This would be of great value for clinicians in selecting drugs likely to be slow in drug resistance emergence, and choose therapies for resistant tumors more effectively. In principle, our microecology technology can be used for many combinations of cancer types and drugs. 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-{kappa}B 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.
关键词：microhabitats ; cancer ; doxorubicin ; evolution ; resistance
