Abstract 715: Mechanism-based evaluation of apoptosis in live tumor cells by triple co-fluorescence staining: Testing algorithmic effectiveness of targeted drugs

Abstract

Abstract Background: Suppression of apoptosis, a morphologically distinct form of cell death is a trait commonly shared by tumor cells which provide them survival advantages and enables them to evolve to drug-resistant states, higher stages, and/or metastasize. Recently we have reported a procedure of Triple co-Fluorescence Staining (FTS) to visualize apoptosis following chemotherapeutic and targeted anti-cancer drugs in live tumor cells (De et al., 2018). Aim: Using triple-co-fluorescence to simultaneously identify 3 crucial mechanisms of apoptosis, (1) enzyme activity of executioner-caspase3, (2) caspase-dependent cell surface exposure of phosphatidylserine and (3) functional mitochondria, we studied apoptosis following combinations of (a) paclitaxel plus BKM120, (b) trametinib plus paclitaxel or CDK4/6 inhibitor in ovarian models, and (c) p110beta inhibitor plus BNM673 in TNBC models. Methods: Ovarian cell lines OVK18, A2780Cis, and SUM149 (TNBC) were used for the study. For the standardization of MitoViewBlue (B) staining, a final concentration of 50 nM was used. Treated live cells on cover-slips were incubated under cultured conditions in the dark for 15-30 minutes with the staining cocktail containing MitoViewBlue (B), NucView® 488 (G) and CF®594 Annexin V (R) in the 1X binding buffer. At the end of the incubation period, pictures were taken (Olympus DP72 digital camera) within a span of 10 minutes. The FTS was corroborated with (1) AnnexinV by flow cytometry, (2) live/dead cell assay, (3) WB expression of apoptotic markers, and (4) mitochondrial depolarization. Results: Using a standardized protocol for FTS we tested apoptosis in ovarian and TNBC models following the combination of chemotherapeutic and targeted anti-cancer drugs. Using B, we stained active mitochondria while using G we stained active caspase3 and R was used to localize plasma membrane asymmetry, flipped phosphatidylserine, a characteristic feature of apoptotic cells. A merged TFS image of live and non-treated (NT) OVK18, A2780Cis and SUM149 cells showed functional mitochondrial stained with bright blue which corroborated with TMRE-based mitochondrial potential. Fewer cells showed membrane fluorescence in red for R and even fewer cells with green fluorescence for G which stained active caspase3. R and G were mutually exclusive. Paclitaxel-treated A2780 cells were significantly devoid of B stains. Membrane blebbing and annexin-V stained cell membrane was identified in cells either with or without G stains but definitely without B stains which indicated the sequence of events of apoptotic in cells. The mutual exclusivity property of B cell to R and/or G cells are preserved as observed with the help of transmission overlay images. Conclusion: TFS can be used to evaluate mechanism-based apoptosis following chemotherapeutic and targeted anti-cancer drugs in live tumor cells. Citation Format: Nandini Dey, Jennifer C. Aske, Casey Williams, Pradip K. De, Brian Leyland-Jones. Mechanism-based evaluation of apoptosis in live tumor cells by triple co-fluorescence staining: Testing algorithmic effectiveness of targeted drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 715.