A Bivalent Activatable Fluorescent Probe for Screening and Intravital Imaging of Chemotherapy‐Induced Cancer Cell Death

Abstract The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high‐resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts in situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real‐time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells in vitro and for direct imaging of chemotherapy‐induced apoptosis in vivo in mouse models of breast cancer.


General experimental information.
Commercially available reactants were used without further purification.

Spectral characterisation. Spectroscopic data was recorded on Cytation 3 (Biotek).
Apotracker Red was dissolved at 5 mM in DMSO and diluted to the indicated concentrations.
Absorbance spectra were recorded on 96-well plates. To determine the relative fluorescence quantum yields, Rhodamine 101 was used as a reference (QY = 0.96 in MeOH). [2] Stability of Apotracker Red in mouse serum. Mouse blood was drawn from 8 week-old C57BL/6 mice into 10% 0.5 M EDTA. Cells were removed by centrifugation for 10 min, 300 g at S19 r.t., followed by separation from erythrocytes by centrifugation for 10 min, 2,000 g at r.t. Sera were transferred into new tubes and stored at -20°C until use. 50 μM Apotracker Red in PBS or in serum were incubated for 24 h at 37 °C on a vertical shaker at 500 rpm. As controls, serum without peptide and freshly aliquoted 50 μM Apotracker Red in mouse serum were used.
Fluorescence spectra were recorded after excitation at 520 nm. For HPLC measurements, proteins were precipitated by addition of CH3CN and centrifugation at 300 g for 5 min at r.t. and the resulting supernatants were injected.

Isolation of neutrophils from human peripheral blood. Ex vivo experiments were performed
with neutrophils isolated freshly isolated from the human peripheral blood of healthy donors.
Work with human peripheral blood leukocytes complied with all relevant ethical regulations and informed consent was obtained. The study protocol was approved by the Accredited Medical Regional Ethics Committee (AMREC, reference number 20-HV-069) at the University of S20 Edinburgh. Human peripheral blood neutrophils were isolated as previously described. [3] Briefly, whole blood was drawn into tubes containing anti-coagulant 3.6% sodium citrate (final concentration: 0.4% (w/v)) and centrifuged at 350 g for 20 min at r.t. with lowest acceleration and no brake. Platelet-rich plasma was removed, and leukocytes separated from erythrocytes by 0.6% dextran sedimentation in saline for 30 min at r.t. The upper layer was further fractionated using an isotonic discontinuous Percoll density gradient. Neutrophils were harvested from the 63% and 72.9% interface and cultured in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) in 5% human AB serum for 18 h at 37°C, 5% CO2 to induce spontaneous apoptosis. Data were acquired using the FACS Diva software and analysed using the FlowJo X software.  Mice were MMTV-PyMT;ACTB-ECFP or MMTV-PyMT;ACTB-ECFP;cfms-EGFP. Tumors were grown to a size between 0.5 and 1 cm in diameter, and then mice were treated with 10 μg g -1 cisplatin. Prior to administration of Apotracker Red, mice were anaesthetized with 4% isofluorane and kept anesthetized under 1-2 % isofluorane and 21% oxygen balanced with nitrogen at 1 L min -1 . Mice were given Apotracker Red (5 μM, 100 μL) intravenously and tumour tissue imaging was performed after 1 h or tissue harvested after 2 h. Mice were positioned on the surgical platform with ventral surface facing up and limbs were secured with laboratory tape.

Induction of apoptosis in
Once the animal was secured, hair was removed using an electronic shaver and chemical hair removal. After disinfection of the ventral surface of the mouse with 70% isopropanol wipes and betadine, mammary gland tumours were exposed by subcutaneous ventral midline incision from about 3 mm above the urethra to the xiphoid process. Skin with the inguinal mammary grand was detached gently from the peritoneal cavity. A microscope slide was positioned against the skin flap and the slide was attached to the external surface of the skin using Krazy Glue. The mouse was positioned on imaging stage in the center of a cover glass-covered imaging point.
Tumours were washed thrice for 10 min with PBS followed by gradual increase with sucrose S23 solution from 12% sucrose to 30% sucrose every 2 h with a 6% higher sucrose solution.
Disposable OCT chambers were used for embedding tumours in OCT on dry ice. OCT solutions were allowed to freeze prior to cutting into 6 μm slides by the Histology Facility of Cold Spring Harbor Laboratories.