Two-photon imaging of intratumoral CD8⁺ T cell cytotoxic activity during adoptive T cell therapy in mice
J. Clin. Invest. Béatrice Breart, et al. 118:1390 doi:10.1172/JCI34388 [Go to this article.]

Figure 4
A fluorescent probe to track tumor cell apoptosis. (A) EG7 tumor cells were stably transfected with a FRET-based fluorescent probe monitoring caspase 3 activity. Briefly, CFP and YFP molecules are linked by a peptide containing the sequence DEVD, which is cleaved by activated caspase 3. EG7 cells were also transfected with a control probe (noncleavable by caspase 3) bearing a mutation in the cleavage motif (DEVG). Cleavage of the probe upon caspase 3 activation resulted in FRET disruption. Tumor cell apoptosis was monitored by 2-photon imaging by calculating the ratio of CFP to YFP emission (for the sake of clarity, this is referred to as the apoptosis index). (B and C) EG7-DEVG or EG7-DEVD tumor cells were subjected to UVB irradiation for 1 minute. Eight hours later, cells were visualized by 2-photon imaging. UVB irradiation resulted in FRET disruption in EG7-DEVD but not in control EG7-DEVG tumors cells. Scale bars: 10 μm. The apoptosis index plotted for individual tumor cells is shown. Tumor cells with a ratio greater than 1.7 were considered to be undergoing apoptosis. (D and E) Flow cytometric analysis of FRET loss in EG7-DEVD and EG7-DEVG tumor cells subjected to UVB irradiation or cocultured with activated OT-I CTLs for 5 hours. The population of EG7 tumor cells displaying FRET loss is shown in green, and the corresponding percentage is indicated.