¹⁸F-ICMT-11 cell uptake correlates to dose-dependent increases in caspase 3 activity following carboplatin treatment.

<p>A: Chemical structure of ¹⁸F-ICMT-11. B: Dose-dependent changes in caspase 3/7 activity following carboplatin treatment. C: Dose-dependent changes in ¹⁸F-ICMT-11 uptake in cells following carboplatin treatment. D: Correlation between caspase 3 activity and ¹⁸F-ICMT-11 uptake in PC9 cells.</p

Voxel-wise analysis of ¹⁸F-ICMT-11 PET imaging data by PVIS.

<p>The intensities of all voxels within the tumour ROIs were computed and expressed as histogram plots of normalized voxel intensity versus the number of voxels. A, B: Typical data from three representative animals (vehicle, 24 h or 48 h carboplatin-treated) for PC9 (A) and A549 (B) are shown. C, D: The statistical comparison of 95^th percentile voxel intensities for PC9 (C) and A549 (D) was performed using Prism v5.0 software (GraphPad). Mean ± SD (<i>n</i> = 4–6 animals per group).*, <i>P</i><0.05; **, <i>P</i><0.01.</p

Differential responses to carboplatin treatment in PC9 and A549 cells.

<p>A: Carboplatin-induced growth inhibition in PC9 and A549 cells using a sulforhodamine B assay 72 h post treatment. B: Western blot analysis of the levels of uncleaved PARP, cleaved PARP and cleaved (active) caspase 3 72 h post carboplatin treatment (0–200 µM) in PC9 and A549 cells. Actin was used as a loading control. C, D: Flow cytometric analysis of PC9 (C) and A549 cells (D) treated with carboplatin (100 µM) or vehicle. Apoptotic cells were identified by Annexin V-Alexafluor488 (λ Ex/Em = 495/519 nm) and necrotic cells by 7-AAD (λ Ex/Em = 546/647 nm). Population Q4 represents viable cells, whereas population Q3 represents apoptotic cells that have low 7-AAD fluorescence and stain with Annexin V. Population Q2 represents secondary apoptotic/necrotic cells.</p

Temporal changes in cell death markers and ¹⁸F-ICMT-11 uptake after carboplatin treatment.

<p>A: Time course of changes in caspase 3/7 activity following carboplatin treatment. B: Western blot analysis of the levels of uncleaved PARP, cleaved PARP and cleaved (active) caspase 3 post 50 µM carboplatin treatment (0–96 h) in PC9 (i) and A549 cells (ii). C: Temporal changes in ¹⁸F-ICMT-11 uptake in cells following carboplatin treatment. D: Correlation between caspase 3 activity and ¹⁸F-ICMT-11 uptake in PC9 cells.</p

Tumour active caspase-3 and TUNEL immunohistochemistry analysis.

<p>Tumour tissues were removed after PET imaging scan, processed for histological analysis and stained for active (cleaved) caspase-3 and DNA fragmentation (TUNEL assay) detection, in conjunction with H&E staining. A: Representative images of histological tumour sections are shown. Staining intensities for cleaved caspase 3 (B) and TUNEL (C) were determined using the ImageJ software and expressed as percent staining per field. Data are mean ± SD. *, <i>P</i><0.05; ***, <i>P</i><0.001. <i>n</i> = 3 tumour sections with 5 random FOV per section. Photographic images of H&E-stained sections were acquired at 100×, with all other images acquired at 400×. Scale bar = 200 µm. Abbreviations: N, necrotic; V, viable.</p

¹⁸F-ICMT-11 PET image analysis of PC9 and A549 xenografts in vehicle and carboplatin-treated mice.

<p>A, B: Tumour volumes recorded by calliper measurements of PC9 (A) and A549 tumours (B) pre- and post-carboplatin treatment as indicated. Data shown are mean ± SD of % volume compared to baseline (<i>n</i> = 4). *, <i>P</i><0.05; **, <i>P</i><0.01. C, D: Representative axial PET-CT images (30–60 min summed activity) for PC9 (C) and A549 (D) tumours. Tumour margins, indicated from CT image, are outlined in red. Mean ± SD (<i>n</i> = 4–6 animals per group). E, F: The tumour TAC representing average counts from a dynamic 60-minute scan for PC9 (E) and A549 xenografts (F) following carboplatin treatment (vehicle, 24 h or 48 h carboplatin-treated; <i>n = </i>4–6 animals per group).</p

Comparison of the C2A Domain of Synaptotagmin-I and Annexin-V As Probes for Detecting Cell Death

The induction of apoptosis is frequently accompanied by the exposure of phosphatidylserine (PS) on the cell surface, which has been detected using radionuclide and fluorescently labeled derivatives of the PS-binding protein, Annexin V. The fluorescently labeled protein has been used extensively in vitro as a diagnostic reagent for detecting cell death, and radionuclide-labeled derivatives have undergone clinical trials for detecting tumor cell death in vivo following treatment. We show here that the C2A domain of Synaptotagmin-I, which had been fluorescently labeled at a single cysteine residue introduced by site-directed mutagenesis, detected the same levels of cell death as a similarly labeled Annexin-V derivative, in drug-treated murine lymphoma and human breast cancer cell lines in vitro. However, the C2A derivative showed significantly less binding to viable cells and, as a consequence, up to 4-fold more specific binding to apoptotic and necrotic cells when compared with Annexin-V. C2A offers a potential route for the development of a new generation of more specific imaging probes for the detection of tumor cell death in the clinic

Supplemental Video M3 from A Systematic Comparison of ¹⁸F-C-SNAT to Established Radiotracer Imaging Agents for the Detection of Tumor Response to Treatment

Supplemental Video M3. 18FML-10 uptake in naive tumors</p

Supplemental Video M4 from A Systematic Comparison of ¹⁸F-C-SNAT to Established Radiotracer Imaging Agents for the Detection of Tumor Response to Treatment

Supplemental Video M4. 18FML-10 uptake in drug-treated tumors</p