title: Screening of breast cancer stem cell inhibitors using a protein kinase inhibitor library.
abstract: BACKGROUND
  Cancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recurrence and metastasis. Based on the characteristic formation of mammospheres in in vitro conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity in breast cancer research. However, manual counting of mammospheres is a time-consuming process that is not amenable to high-throughput screening, and there are occasional inaccuracies in the process of determining the mammosphere diameter. In this study, we proposed a novel automated counting method of mammosphere using the National Institute of Standards and Technology (NIST)'s Integrated Colony Enumerator (NICE) with a screening of protein kinase library.
  
  
  METHODS
  Human breast cancer cell line MCF-7 was used for evaluation of tumor sphere efficiency, migration, and phenotype transition. Cell viability was assessed using MTT assay, and CSCs were identified by an analysis of CD44 expression and ALDEFLUOR assay using flow cytometry. Automated counting of mammosphere using NICE program was performed with a comparison to the result of manual counting. After identification of inhibitors to ameliorate CSC formation by screening a library of 79 protein kinase inhibitors using automated counting in primary, secondary and tertiary mammosphere assay, the effect of selected kinase inhibitors on migration, colony formation and epithelial-to-mesenchymal transition (EMT) of MCF-7 cells was investigated.
  
  
  RESULTS
  Automated counting of mammosphere using NICE program was an easy and less time-consuming process (<1 min for reading 6-well plate) which provided a comparable result with manual counting. Inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII), Janus kinase-3 (JAK-3), and IκB kinase (IKK) were identified to decrease the formation of MCF-7-derived CSCs in primary, secondary and tertiary mammosphere assay. These protein kinase inhibitors alleviated TGF-β1-induced migration, colony formation and EMT of MCF-7 cells.
  
  
  CONCLUSIONS
  We have developed a novel automated cell-based screening method which provided an easy, accurate and reproducible way for mammosphere quantification. This study is the first to show the efficacy of an automated medium-throughput mammosphere-counting method in CSC-related research with an identification of protein kinase inhibitors to ameliorate CSC formation.
authors.affiliation: The Division of Nephrology, Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul, 07985 Republic of Korea.
authors.forename: Hack Sun
authors.initials: HS
authors.lastname: Choi
authors.affiliation: The Division of Nephrology, Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul, 07985 Republic of Korea.
authors.forename: Dal-Ah
authors.initials: DA
authors.lastname: Kim
authors.affiliation: Department of Life Science, The Research Center for Cellular Homeostasis, Ewha Womans University, Seoul, 03760 Republic of Korea.
authors.forename: Heesung
authors.initials: H
authors.lastname: Chung
authors.affiliation: Department of Microbiology, Yonsei University College of Medicine, Seoul, 03722 Republic of Korea.
authors.forename: In Ho
authors.initials: IH
authors.lastname: Park
authors.affiliation: Division of Translational and Clinical Research II, Hematologic Malignancy Branch, National Cancer Center, Goyang-si, Gyeonggi-do 10408 Republic of Korea.
authors.forename: Bo Hye
authors.initials: BH
authors.lastname: Kim
authors.affiliation: Department of Life Science, The Research Center for Cellular Homeostasis, Ewha Womans University, Seoul, 03760 Republic of Korea.
authors.forename: Eok-Soo
authors.initials: ES
authors.lastname: Oh
authors.affiliation: The Division of Nephrology, Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul, 07985 Republic of Korea.
authors.forename: Duk-Hee
authors.identifier: 0000-0001-8475-8932
authors.initials: DH
authors.lastname: Kang
keywords: Breast cancer; Cancer stem cell; Mammosphere; Protein kinase inhibitor