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.


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Electronic Supporting Information
Purification was conducted by semi-preparative HPLC using a 0-100% gradient over 25 min, with detection at 560 nm.Pure fractions were collected and lyophilised to afford pure Apotracker Red as a purple solid (4.0 mg, 2.9 μmol, 22% overall yield).

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]ability 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. 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.Medium supplemented with 10% fetal bovine serum (FBS), 100 U mL -1 penicillin and 0.1 mg mL -1 streptomycin.A549 and PC3 cells were cultured in Dublecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U mL -1 penicillin and 0.1 mg mL -1 streptomycin.

Cell
Flow cytometry drug screen.To evaluate the proliferation of cancer cells, labelling with CellTrace TM Violet was performed according to the manufacturer's instructions.Briefly, cells were resuspended at 6×10 6 cells mL -1 and incubated for 30 min with 1 μM CellTrace TM Violet.
On the following day, drugs were dissolved in DMF at 10 mM, diluted to the indicated working concentrations and incubated with the cells for 24 or 48 h, as indicated.On the final day, cells S21 were centrifuged (300 g, 5 min, r.t.), washed once with PBS before addition of 0.05% Trypsin, 0.02% ethylenediaminetetraacetic acid (EDTA) in Hank's balanced salt solution (HBSS) and incubation at 37°C for 5 min.Detached cells were combined in 96-well round bottom plate followed by Apotracker Red staining.Histograms and mean fluorescence intensities of apoptotic cells were measured on a 5L LSR flow cytometer under the following excitation/emission filters: Apotracker Red (561/610 nm), Annexin V-AF647 (647/670 nm).
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.

Figure S16 .Figure S17 .
Figure S16.Quantification of EGFP + macrophages in tumours from untreated and cisplatin- t. and the resulting supernatants were injected.Lipid layer assays.Cardiolipin (CL), phosphatidylglycerol (PG) and phosphatidic acid (PA) were purchased from Stratech Scientific Ltd.Phosphatidylserine (PtdSer) and phosphatidylcholine (PC) were obtained from Sigma Aldrich.All lipids were dissolved at 1 mg mL -1 in dry EtOH by vigorous vortexing for 5 min followed by sonication for 20 min.100 μL of the lipids were then transferred into a black flat-bottom 96-well plate to generate lipid layers by solvent evaporation at 20 o C overnight in a sterile fume hood.Apotracker Red was reconstituted at 1 μM in sterile PBS, added to the wells and incubated at 25 o C for 40 min.The limit of detection (LoD) of Apotracker Red for PtdSer was determined by fluorescence titration of serial dilutions of PtdSer layers after incubation with Apotracker Red (1 μM, PBS) at 25 o C for 40 min.The LoD was calculated using the equation LoD = (3 × σ)/k, where σ is the standard deviation of blank solutions and k is the slope of the linear regression fit.Data was recorded on Cytation 3 (exc: 530 nm).
culture.MCF-7, A549, LnCap, MDA-MB-231, Jurkat T cells and PC3 cells were obtained from American Type Culture Collection (ATCC).HT29 cells were purchased from Merck (Sigma Aldrich, UK).Mouse Mammary Tumour Virus-Polyoma Middle T antigen (MMTV-PyMT) cells were provided by the lab of Mikala Egeblad (CSHL, US).MMTV-PyMT, MCF-7, LnCap, MDA-MB-231 and Jurkat T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Jurkat T cells.Jurkat T cells were induced to undergo apoptosis using 1 μM staurosporine.For confocal imaging, cells were resuspended in RPMI supplemented with 10% FBS, 100 U mL -1 penicillin and 0.1 mg mL -1 streptomycin and 2.5×10 5 per well.Cells were imaged between 1 and 3 h after induction of apoptosis and flow cytometry experiments were performed after 2 h of treatment.Live-cell fluorescence microscopy.Live-cell imaging was performed either on a TCS SP8 confocal microscope (Leica) or in a spinning-disk confocal microscope (Andor).For floating cells, 2.5×10 5 cells per well were plated in Nunc TM Lab-Tek TM II 8-well chamber slides and allowed to settle for 30 min prior to imaging.For adherent cells, cells were plated at 5×10 4 cells per well in Nunc TM Lab-Tek TM II 8-well chamber slides on the day prior to imaging.Nuclear staining was performed for 20 min at r.t. with 7 μM Hoechst 33342 prior to imaging together with 150 nM Apotracker Red and/or 5 nM Annexin V-AF647.Hoechst 33342 (exc: 405 nm), Apotracker Red (exc: 561 nm), Annexin V-AF647 (exc: 633 nm).MMTV-PyMT breast cancer mouse model and intravital imaging of cisplatin-induced apoptosis.The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Cold Spring Harbor and performed at the Cold Spring Harbor Laboratory S22 Animal Shared Resources.Animal testing and research complied with the NIH Guide for Care and Use of Laboratory Animals.Mice were housed in a specific-pathogen-free facility with standard husbandry, temperature 19-22 °C, humidity 45-55%.Dark and light cycles were 12 h.