Abstract
BackgroundCancer 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.MethodsHuman 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.ResultsAutomated 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.ConclusionsWe 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.
Highlights
Cancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recur‐ rence and metastasis
We proposed the method of mediumthroughput and automated mammosphere counting using publicly available colony counting software, the National Institute of Standards and Technology (NIST)’s Integrated Colony Enumerator (NICE) [6]
Formation of mammospheres from MCF‐7 cells An incubation of MCF-7 cells with complete MammoCultTM medium resulted in the formation of spherical mammospheres in 7 days (Fig. 1a)
Summary
Cancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recur‐ rence and metastasis. CSCs are generally defined by self-renewal characteristics and their ability to differentiate into non-stem cancer cells [1]. Another important characteristic of CSCs is the resistance to conventional chemotherapy and radiotherapy, which causes a drug-resistance and relapse [2]. In addition to cell sorting-based CSC identification method, marker-specific tracking of subpopulations of cancer cells in competitive tumor models has recently been used to further establish in vitro and in vivo evidence for the presence of CSCs. CSCs have distinct markers allowing for their isolation based on the expression of specific proteins, which includes CD44, CD133, Oct, Sox, Nanog, ALDH1, ABCG, and CXCL12 [3]. The standard methods for assaying CSC activity are in vivo transplantation and in vitro tumorsphere formation [4]
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