Multiplexed apoptosis targeting.

Apoptosis is an important mechanism in cellular homeostasis. Imbalances in the apoptotic process are associated with various disease states.

An important example is the acquired ability of cancer cells to resist their own programmed cell death and therefore it is the aim of many tumor therapies to either reestablish pro-apoptotic signaling pathways or induce apoptosis through activation of existing mechanisms within the cell.

Therefore, visualizing and quantifying the apoptotic process in vivo has great value in monitoring therapy response, diagnosis and staging disease.

Visualization of apoptosis probe accumulation in hypoxic tumor regions

Panel A shows the single-wavelength (760 nm) anatomical optoacoustic image of the tumor region in a Balb/c nu/nu mouse with an orthotopic 4T1 mammary tumor. The tumor cells were implanted in the right abdominal mammary fat pad and allowed to grow for 10 days; the dashed line outlines the tumor margin. Panel B and C show spectrally unmixed, pseudo-colored signals for oxygenated and deoxygenated hemoglobin, respectively. A more hypoxic region is readily identified in the overlay image (panel D) by the purple color. Panel E shows the signal resulting from DyLight 747-conjugated caspase probe in the Jet color-scheme overlain on a single-wavelength (900 nm) anatomical optoacoustic image. Maximal apoptosis signal is clearly co-localized with more hypoxic regions in the tumor.

Simultaneous quantification of apoptosis probe and control dye dynamics

DyLight 747-conjugated apoptosis probe was systemically co-injected with DyLight 690 control dye into 4T1 tumor bearing mice. Panel A shows the quantification of the signal from apoptosis probe (green) and the control dye (blue) over time. Panel B shows a z-stack of cross-sectional images of signals from each probe at T = 60 min. using the same color coding. Panel C shows a corresponding ex vivo cryoslice, showing fluorescent signal of each probe on a background color image. In both the MSOT (B) and cryo-fluorescence (C) images, control dye and apoptosis probe are both detected in the abdominal area, but in the tumor area strong signals are only present from the apoptosis probe.

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