High-Throughput Drug Screening Revealed That Ciclopirox Olamine Can Engender Gastric Cancer Stem-like Cells.

Cancer stem cells (CSCs) are relevant therapeutic targets for cancer treatment. Still, the molecular circuits behind CSC characteristics are not fully understood. The low number of CSCs can sometimes be an obstacle to carrying out assays that explore their properties. Thus, increasing CSC numbers via small molecule-mediated cellular reprogramming appears to be a valid alternative tool. Using the SORE6-GFP reporter system embedded in gastric non-CSCs (SORE6-), we performed a high-throughput image-based drug screen with 1200 small molecules to identify compounds capable of converting SORE6- to SORE6+ (CSCs). Here, we report that the antifungal agent ciclopirox olamine (CPX), a potential candidate for drug repurposing in cancer treatment, is able to reprogram gastric non-CSCs into cancer stem-like cells via activation of SOX2 expression and increased expression of C-MYC, HIF-1α, KLF4, and HMGA1. This reprogramming depends on the CPX concentration and treatment duration. CPX can also induce cellular senescence and the metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis. We also disclose that the mechanism underlying the cellular reprogramming is similar to that of cobalt chloride (CoCl2), a hypoxia-mimetic agent.

A SOX2 Reporter System Identifies Gastric Cancer Stem-Like Cells Sensitive to Monensin.

Gastric cancer remains a serious health burden with few therapeutic options. Therefore, the recognition of cancer stem cells (CSCs) as seeds of the tumorigenic process makes them a prime therapeutic target. Knowing that the transcription factors SOX2 and OCT4 promote stemness, our approach was to isolate stem-like cells in human gastric cancer cell lines using a traceable reporter system based on SOX2/OCT4 activity (SORE6-GFP). Cells transduced with the SORE6-GFP reporter system were sorted into SORE6+ and SORE6- cell populations, and their biological behavior characterized. SORE6+ cells were enriched for SOX2 and exhibited CSC features, including a greater ability to proliferate and form gastrospheres in non-adherent conditions, a larger in vivo tumor initiating capability, and increased resistance to 5-fluorouracil (5-FU) treatment. The overexpression and knockdown of SOX2 revealed a crucial role of SOX2 in cell proliferation and drug resistance. By combining the reporter system with a high-throughput screening of pharmacologically active small molecules we identified monensin, an ionophore antibiotic, displaying selective toxicity to SORE6+ cells. The ability of SORE6-GFP reporter system to recognize cancer stem-like cells facilitates our understanding of gastric CSC biology and serves as a platform for the identification of powerful therapeutics for targeting gastric CSCs.

Collar occupancy: A new quantitative imaging tool for morphometric analysis of oligodendrocytes.

BACKGROUND: Oligodendrocytes (OL) are the myelinating cells of the central nervous system. OL differentiation from oligodendrocyte progenitor cells (OPC) is accompanied by characteristic stereotypical morphological changes. Quantitative imaging of those morphological alterations during OPC differentiation is commonly used for characterization of new molecules in cell differentiation and myelination and screening of new pro-myelinating drugs. Current available imaging analysis methods imply a non-automated morphology assessment, which is time-consuming and prone to user subjective evaluation. NEW METHOD: Here, we describe an automated high-throughput quantitative image analysis method entitled collar occupancy that allows morphometric ranking of different stages of in vitro OL differentiation in a high-content analysis format. Collar occupancy is based on the determination of the percentage of area occupied by OPC/OL cytoplasmic protrusions within a defined region that contains the protrusion network, the collar. RESULTS: We observed that more differentiated cells have higher collar occupancy and, therefore, this parameter correlates with the degree of OL differentiation. COMPARISON WITH EXISTING METHODS: In comparison with the method of manual categorization, we found the collar occupancy to be more robust and unbiased. Moreover, when coupled with myelin basic protein (MBP) staining to quantify the percentage of myelinating cells, we were able to evaluate the role of new molecules in OL differentiation and myelination, such as Dusp19 and Kank2. CONCLUSIONS: Altogether, we have successfully developed an automated and quantitative method to morphologically characterize OL differentiation in vitro that can be used in multiple studies of OL biology.