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Cancer cells require the regulation of organelle-specific unfolded protein responses, such as endoplasmic reticulum (ER) stress, because of their increased metabolic activity during rapid proliferation and cell growth, which are executed through the activation of diverse signaling pathways. In this study, we focused on the dynamic regulation of ER stress in accordance with cancer cellular demand, and we performed real-time monitoring of the activation of the binding immunoglobulin protein (Bip) promoter, which is one of the most responsive genes to ER stress during cancer cell growth, in two and three dimensional (2D and 3D) cell culture using bioluminescence imaging at the single-cell level. Bioluminescence images were obtained from living single cancer cells after transient transfection of the reporter gene, and we observed Bip promoter activation during cell growth. Bip promoter activation was also observed in 2D and 3D culture using stably transfected glioblastoma cancer cells with the reporter gene. The Bip promoter was activated especially in dividing cells during cell growth. We then performed real-time monitoring of Bip promoter activation by bioluminescence imaging in tissue slices obtained from U251/pBipPro-Luc tumors. Luminescence intensity was not constant and was different in individual regions of the tumor slices, and the Bip promoter was activated in several regions during monitoring in vitro. These results show that real-time monitoring by bioluminescence imaging at the single-cell level is a suitable tool for not only gene analysis of signal transduction and regulation of the dynamics of the unfolded protein response in cancer cells but also for the evaluation of the efficacy of anti-cancer agents, and could provide additional information that has been difficult to obtain using conventional assays. Abbreviations: ER: Endoplasmic Reticulum, 2D or 3D: Two or Three Dimensional, Bip/GRP78: Binding Immunoglobulin Protein/78 kDa Glucose-Regulated Protein, LV200: LUMINOVIEW LV200, EGFR: Epidermal Growth Factor Receptor, Akt: Protein Kinase B, PBS: Phosphate-Buffered Saline, ROI: Region of Interest

Monitoring Bip promoter activation during cancer cell growth by bioluminescence imaging at the single-cell level