Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes

Abstract

A long-term goal of cancer diagnosis is to develop tumor-imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from nontarget tissues is crucial. Here we achieve highly specific in vivo cancer visualization by using a newly designed targeted 'activatable' fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH-activatable probes based on the boron-dipyrromethene fluorophore were synthesized and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex vivo and in vivo imaging of human epidermal growth factor receptor type 2-positive lung cancer cells in mice was performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific, cell surface-targeting molecules that result in cellular internalization.

Figure 1. Development of a series of fluorescence probes for various acidic environments

(a) A scheme for the reversible and acidic pH-induced fluorescence activation of a probe. (b) pH profiles of fluorescence of H₂NBDP, DiMeNBDP, EtMeNBDP and DiEtNBDP as acidic pH-sensitive fluorescence probes, and PhBDP as a control “always on” probe. The pH ranges from pH 2 (left) to pH 9 (right) in one pH unit increments. (c) pH dependent changes in emission intensity of acidic pH-activatable BODIPY probes. Curve fitting was based on a modified Henderson-Hasselbach equation. (d) pH-dependent changes in emission intensity of acidic pH-activatable BODIPY-trastuzumab conjugates around the physiological pH range. DOL: PhBDP-IgG = 3.0, DiEtNBDP-IgG = 2.8, EtMeNBDP-IgG = 2.8, DiMeNBDP-IgG = 2.7

Figure 2. In vitro imaging of NIH3T3/HER2 cells with PhBODIPY-, DiMeNBODIPY-, EtMeNBODIPY- or DiEtNBODIPY-labeled trastuzumab

(a) Confocal microscope images of NIH3T3/HER2 cells obtained just after the addition of the probes, and at 2, 4, and 24 hours post-addition of BDP-conjugated trastuzumab to the medium. (b) A solution of DiEtNBDP-, or PhBDP-conjugated Trastuzumab was added to NIH3T3/HER2 cells. Following incubation for 24 hr, LysoTracker Red DND-99 was added, and fluorescent images were obtained 1 hr post-addition of the organelle marker.

Figure 3. The activatable probe can produce a fluorescence signal only from HER2-positive tumors and not from HER2-negative tumors with minimal background from normal tissue

(a) White light (upper panel), spectrally unmixed green fluorescence (middle panel) and composite overlapped images (lower panel) of the lung one day after injection of always on (left) and activatable (right) optical imaging probes are shown. The activatable probe produces a fluorescence signal only from tumors in the lung, whilst the control “always on” probe produces a fluorescence signal not only from tumors, but also from the background normal lung and heart. The findings were observed consistently in all 8 sets of lungs examined. The first 3 consecutive sets of lungs are shown in the figure. (b) The activatable probe (bottom right) readily distinguishes HER2+ (green) and RFP+/HER2− (red) tumors in the lung. In contrast, the control probe produces a green fluorescence signal not only from HER2+ tumors, but also from the normal background tissue and the RFP+/HER2− tumors - seen as yellow (a mixture of the green and red colors). Upper panel; White light images, Lower panel; spectrally unmixed and composite multicolor fluorescent images. (c) In the same mouse model as shown in (b), HER2-specific pH-activatable probe (trastuzumab; left) demonstrated HER2+ (green) and RFP+/HER2− (red) tumors in the lung, however, control pH-activatable humanized antibody (daclizumab, right) did not reveal any tumors. The normal tissue does not demonstrate fluorescence with either probe.

Figure 4. The activatable probe can produce a fluorescence signal only from living HER2-positive cells and tumors, and not from dead cells or fixed tumors

(a) Fluorescence and bright light images of NIH3T3/HER2 cells with PhBODIPY-, DiMeNBODIPY-, EtMeNBODIPY- or DiEtNBODIPY-labeled trastuzumab. Fluorescence images were obtained 5 hr after addition of the labeled trastuzumab (left), and then after treatment with 30% ethanol followed by 10-min incubation (right). (b) White light (left panels) and spectrally unmixed and composite fluorescence (right panels) images of lungs one day after i.v. injection of “always on” (left: white arrows) and activatable (right: yellow arrows) optical imaging probes and pretreatment (upper panels) or 30 min after dipping in the 100% ethanol (lower panels) are shown. The fluorescence signals from most of the small tumors, visualized with the activatable probe (upper right) became almost invisible 30 min after dipping in 100% ethanol (lower right), while the fluorescence signals from most of the tumors loaded with the “always on” probe (upper left), were essentially unchanged. (c) Signal changes of individual surface tumors of 1.0 ± 0.3 mm in size before and after alcohol treatment were plotted. The fluorescence signals from most of the small tumors, which were visualized with the activatable probe (right), showed significant decrease in signal compared with those with the “always on” probe (P < 0.01).

Scheme 1

(a) Existing strategies for tumor imaging with MRI, PET or non-activatable “always on” fluorescence detection. (b) Novel strategy for selective tumor imaging with activatable fluorescence probes. (c) A schematic representation of highly selective tumor imaging with an activatable fluorescence probe-antibody conjugate. The probe is non-fluorescent when outside the tumor cells. Following internalization by the endocytosis, the probe is accumulated in late endosomes or lysosomes, where the acidic pH activates the probe, making it highly fluorescent.