Steps in Semiautomated Image Analysis

<p>Semiautomated image analysis involves recognition and automated segmentation of each lymph node (A), quantitation of magnetic tissue parameters (T2*, variance of pixel values; [B]), comparison of extracted tissue parameter to a database (C), and 3D reconstruction of nodal anatomy onto vascular anatomy (D).</p

Breast Cancer Mapping

<div><p>Patient with breast cancer prior to sentinel lymph node biopsy.</p> <p>(A) Conventional axillary MRI shows nonenlarged lymph nodes that do not meet the size criteria of malignancy (white bar = 5 mm).</p> <p>(B) Following intravenous administration of nanoparticles, a single 3-mm intranodal metastasis was correctly identified.</p> <p>(C) Ex vivo MRI of sentinel node specimen confirms metastasis.</p> <p>(D) Semiautomated nodal analysis and reconstruction correctly juxtaposed solitary lymph node metastases adjacent to two normal lymph nodes.</p> <p>(E) Correlative histopathology confirms the diagnosis. For 3D reconstruction of axillary nodal anatomy see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0010066#pmed-0010066-s002" target="_blank">Video S2</a>.</p></div

Tissue Parameters in Learning Dataset

<p>Nodal tissue parameters for benign and malignant nodes are shown before (A and B) and after (C–E) intravenous administration of magnetic nanoparticles. Note the insensitivity of conventional MRI (A and B), better separation using single-value analysis (C and D) and excellent separation using two-value analysis (E).</p

<b>A:</b> Short axis high resolution, high field cardiac MRI of a FXIII^−/− mouse 2 days after coronary ligation.

<div><p>Arrows: intrathoracic hematoma adjacent to experimental anterolateral infarction.</p> <p> <b>B:</b> Autopsy confirms a blood clot (asterisk) originating from myocardial rupture at the border zone (arrow) of the myocardial infarct.</p> <p> <b>C:</b> Histology of 1A shows rupture channel (arrows), filled with blood.</p> <p> <b>D:</b> In patients with ruptured MI, FXIII levels were significantly reduced (*p<0.01).</p> <p> <b>E:</b> Color Doppler echo of patient with new ventricular septum defect 7 days after myocardial infarction (arrow).</p> <p> <b>F:</b> MRI after VSD repair with patch (arrows).</p> <p> <b>G–I:</b> Explantation site of saphenous veins for CABG surgery displays delayed healing.</p> <p> <b>J:</b> 73 days after initial surgery, 3 revisions and 2 weeks after i.v. FXIII augmentation, the wound is closed.</p></div

Bioorthogonal labeling using the specific MET CID PF04217903-TCO (15) or the PCID Foretinib-TCO (11) in OVCA429 (MET positive) and SK-BR-3 (MET negative) cells.

<p>Cells were incubated for 1hr with 200 nM Foretinib-TCO (<b>11</b>) or 40 nM PF04217903-TCO (<b>15</b>), washed and incubated for 30 min with 1 μM Tz-CFDA (e-h) for bioorthogonal reaction inside living cells. After fixation with 2% paraformaldehyde, MET was labeled using a MET primary antibody and AlexaFluor 647 labeled secondary antibody (i-l). After nuclear staining with Hoechst 33342 (a-d) for 10 min, 40X images were collected using a DeltaVision microscope. Note the excellent co-localization between the MET antibody and affinity ligands on the membrane of the cells (m-p). Scale bar: 10 μm.</p

Crystal structure of MET (gray) showing the design of the bioorthogonal MET imaging agents.

<p>(A) Foretinib-TCO (B) PF04217903-TCO. Note that the bioorthogonal transcyclooctene (TCO) is predicted to project outside from the target so that it is available for reaction with the fluorescent counter partner. (PDB ID: 3LQ8 and 3ZXZ) 3D models were rendered using PyMol.</p

Synthetic scheme of Foretinib-TCO (11), Foretinib-BODIPY-FL (12) and PF04217903-TCO (15).

<p>Boc = <i>tert</i>-butyloxycarbonyl; Bn = Benzyl; BODIPY-FL = 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl; DMAP = 4-dimethylaminopyridine; DCM = dichloromethane; DIPEA = N,N-Diisopropylethylamine; DME = dimethoxyethane; DMF = dimethylformamide; Ms = methanesulfonyl; TCO = trans-cyclooctene; TEA = triethylamine; TFA = trifluoroacetic acid; THF = tetrahydrofuran; NHS = <i>N</i>-hydroxysuccinimide.</p

Inhibitory effect of Foretinib based imaging agents.

<p>(A) The IC₅₀ values for Foretinib, Foretinib-TCO (<b>11</b>) and Foretinib-BODIPY-FL (<b>12</b>) against purified MET were determined using the z’-lyte kinase assay. Note the much lower affinity of the fluorochrome conjugated drug compared to the bioorthogonal version. (B) Representative western blot of MET phosphorylation inhibition by Foretinib, Foretinib-TCO (<b>11</b>) and Foretinib-BODIPY-FL(<b>12</b>) in OVCA429 cells. Following pre-treatment with increasing concentrations of inhibitors (0, 100, 500, or 1000 nM, respectively), MET phosphorylation was stimulated with HGF for 10 min, followed by cell lysis, SDS-PAGE, and Western blot with MET and phospho-MET antibodies. (C) Densiometric quantification of MET phosphorylation from the Western blot data using ImageJ. (D) The IC₅₀ values for Foretinib and Foretinib-TCO (<b>11</b>) against purified AXL, PDGFRα, RON and KDR were determined using the z’-lyte kinase assay. R² values for the dose-response curve fit (GraphPad, Prism) were 0.92 or greater.</p

Analysis of average nuclear drug concentrations over time.

<p>A. Representative images from a 5 hour PARP inhibitor pharmacokinetics assay. Far-Left Panel: drug distribution. Scale bar represents 50 µm. Middle-Left Panel: H2B Nuclear Marked tumor cells. Scale bar represents 50 µm. Middle-Right Panel: merged images displaying both the drug (green) and tumor cells (red). An area of the closest vessel was also selected to analyze the dynamics of drug distribution through the vasculature (Red box). Scale bar represents 50 µm. Far-Right Panel: Magnified cells from the presented image shown over time. The white arrow indicates a single cell visually tracked throughout the course of the video. Scale bar represents 10 µm. B. The average and standard deviation of nuclear drug concentration in all cells over time was analyzed using the described segmentation algorithm. The vessel concentration dynamics were also analyzed by quantifying drug channel fluorescence within an area of the vessel. C. The number of cells receiving a therapeutic dose of the drug over time.</p

Single Cell Pharmacokinetic Tracking.

<p>The segmentation algorithm was combined with a linking program to determine individual cell nuclear drug concentrations. A. The locations of cell nuclei were tracked over 5 hours, using external linking software in a video where both cell movement and image drift were present. Red boxes indicate arbitrarily selected cells used for manual tracking verification of the algorithm. B. Manual tracking of arbitrarily selected cells. C. By combining results using the segmentation algorithm together with the tracking data, drug concentration over time in 10 sample cells could be plotted.</p