Intravital FRET Imaging of Tumor Cell Viability and Mitosis during Chemotherapy
Figure 3
Docetaxel increases caspase-3 activity in vitro and in vivo.
A, quantification of the CFP-YFP ratio of SW480 cells in vitro under indicated conditions (1 µm docetaxel). The 24* indicates that only apoptotic cells were analyzed, in all other cases (0, 16, and 24) mitotic cells were quantified. Apoptotic and mitotic cells were determined according to morphology. Apoptotic: membrane blebbing, fragmented DNA. Mitotic: condensed DNA, rounded membrane morphology. 10 cells were quantified per condition. Average + SEM is shown. B, quantification of the CFP-YFP ratio upon intravital imaging of tumor cells stably expressing caspase-3 FRET sensor. The CFP-YFP ratios (normalized to average CFP-YFP value before treatment) of individual cells in vivo of the same photoswitched tumor fields were plotted against the indicated times after a single intravenous injection of 25 mg/kg docetaxel. Red dotted line represents the average normalized CFP-YFP ratio of vehicle (PBS)-treated mice as shown in Figure S3A. Results of 3 independent experiments (visualized by different symbols for different mice) per cell line are shown. One symbol represents one cell. Line indicates median + IQR. ***: significant (Mann Whitney U test, p<0.008). C, left: Color scheme of CFP fluorescence lifetime analysis as explained in Figure 2A and C. Short lifetimes (blue) indicate high FRET levels, long lifetimes (red) indicate low FRET levels. Right: Representative FLIM images of a C26 tumor showing a group of tumor cells before (−) (upper panel) and 20 hours after (+) (lower panel) 25 mg/kg docetaxel treatment. D, the fluorescent lifetime of C26 cells in vivo plotted at indicated time points after docetaxel treatment. The red dotted line represents the average lifetime of vehicle (PBS)-treated mice as shown in Figure S3B. One representative experiment is shown of two independent experiments. One dot represents one cell. ***: significant (Mann Whitney U test, p<0.0001). Line indicates median + IQR.