Reproducibility of sequential images in MCF7-mCherry-luc tumors.

<p>A group of nine MCF7-mCherry-luc tumors was repeatedly observed by FLI, BLI, and PD US following injection of 100 µl saline IP. A) Sequential FLI for a representative mouse, B) BLI from the same mouse showing images acquired 10 mins after administration of fresh luciferin on each occasion, c) PD US showing MIP (maximum intensity projection) observed at 40 MHz, D) Variation in dynamic bioluminescent signal intensity from the same tumor as in A,B and C, on five sequential occasions over 24 hrs, E) Comparison of signal stability based on FLI (red <b>□</b>), BLI (blue ▴) and PD-US (black ▴) normalized values are presented mean ± SE for the whole group.</p

In Vivo Chemiluminescent Imaging Agents for Nitroreductase and Tissue Oxygenation

Tissue oxygenation is a driving parameter of the tumor microenvironment, and hypoxia can be a prognostic indicator of aggressiveness, metastasis, and poor response to therapy. Here, we report a chemiluminescence imaging (CLI) agent based on the oxygen-dependent reduction of a nitroaromatic spiroadamantane 1,2-dioxetane scaffold. <b>Hy</b>poxia <b>C</b>hemi<b>L</b>uminescent Probe <b>2</b> (<b>HyCL-2</b>) responds to nitroreductase with ∼170-fold increase in luminescence intensity and high selectivity for enzymatic reductase versus other small molecule reductants. <b>HyCL-2</b> can image exogenous nitroreductase in vitro and in vivo in living mice, and total luminescent intensity is increased by ∼5-fold under low oxygen conditions. <b>HyCL-2</b> is demonstrated to report on tumor oxygenation during an oxygen challenge in H1299 lung tumor xenografts grown in a murine model as independently confirmed using multispectral optoacoustic tomography (MSOT) imaging of hemoglobin oxygenation

Optical assessment of vascular disruption in U87-mCherry-luc tumors.

<p>Optical imaging was performed at various times before and after administration of ATO. On each occasion FLI was performed first and then <i>D</i>-luciferin was administered SC in the neck and BLI was performed over a period of 16 mins. <b>A</b>) Sequential FLI with respect to a dose of 8 mg/kg ATO administered IP, <b>B</b>) Corresponding time course of bioluminescent signal evolution in this mouse following luciferin injection at respective times, <b>C</b>) BLI signal intensity at the 10 minute time point following luciferin administration, <b>D</b>) Normalized BLI signal intensity at 10 minute time point after administration of luciferin indicating vascular shutdown following various doses of ATO.</p

Dual ¹⁹F/¹H MR Gene Reporter Molecules for <i>in Vivo</i> Detection of β-Galactosidase

Increased emphasis on personalized medicine and novel therapies requires the development of noninvasive strategies for assessing biochemistry <i>in vivo</i>. The detection of enzyme activity and gene expression <i>in vivo</i> is potentially important for the characterization of diseases and gene therapy. Magnetic resonance imaging (MRI) is a particularly promising tool, since it is noninvasive and has no associated radioactivity, yet penetrates deep tissue. We now demonstrate a novel class of dual ¹H/¹⁹F nuclear magnetic resonance (NMR) <i>lacZ</i> gene reporter molecule to specifically reveal enzyme activity in human tumor xenografts growing in mice. We report the design, synthesis, and characterization of six novel molecules and evaluation of the most effective reporter in mice <i>in vivo</i>. Substrates show a single ¹⁹F NMR signal and exposure to β-galactosidase induces a large ¹⁹F NMR chemical shift response. In the presence of ferric ions, the liberated aglycone generates intense proton MRI T₂ contrast. The dual modality approach allows both the detection of substrate and the imaging of product enhancing the confidence in enzyme detection

Vascular disruption assessed by BLI and PD-US in MCF7-mCherry-luc tumors with respect to ATO (8 mg/kg).

<p>A) Variation in bioluminescent signal intensity from a representative tumor on sequential occasions over 24 hrs: blue ♦ baseline; red ▪ 4 hrs; green ▴ 24 hrs. B) BLI acquired 10 mins after administration of luciferin. C) PD US images are presented as single slice (left) and PD maximum intensity projection (right) before and 4 hrs after treatment with ATO. D) Comparison of PD US and BLI in MCF7-mCherry-luc tumors as fractional signal versus baseline.</p

Histology showing vascular impairment and apoptosis in MCF7-mCherry-luc tumors.

<p>Sections were obtained from a series of mice sacrificed at various times after ATO (8 mg/kg). Hoechst stain shows reduced perfusion 2–6 hrs following ATO and H&E stain shows increased necrosis after 24 hrs. Left column: vascular extent (CD31; green) and perfusion (Hoechst 33342; blue). Middle column: Caspase-3 activity indicating apoptosis. Right column: H&E.</p

Optical detection of U87-mCherry-luc tumor growth.

<p>A) FLI (λ_Ex = 570 nm and λ_Em = 620 nm) showed tumor growth on the back of a nude mouse. B) Corresponding BLI acquired 10 mins after administration of sodium <i>D</i>-luciferin (80 µL, 40 mg/ml). C) Correlation between signal intensities and caliper measured tumor volume for a group of six U87-mCherry-Luc tumors; FLI (•; R²>0.82) and BLI (Δ; R²>0.86); D) Correlation between FLI and BLI photon signal intensities at each measurement time (R²>0.86).</p

Comparative vascular shutdown following ATO (8 mg/kg) to mice bearing various tumors.

<p>Relative BLI signal intensity observed for tumors 10 mins after administration of luciferin with respect to drug treatment in three different types: blue ♦ MCF7-mCherry-luc; green ▴U87-mCherry-luc; red ▪ PC3-luc; mean values ± SE.</p

Tubulin-Destabilizing Agent BPR0L075 Induces Vascular-Disruption in Human Breast Cancer Mammary Fat Pad Xenografts

<div><p>BPR0L075, 6-methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1<em>H</em>-indole, is a tubulin-binding agent that inhibits tubulin polymerization by binding to the colchicine-binding site. BPR0L075 has shown antimitotic and antiangiogenic activity <em>in vitro</em>. The current study evaluated the vascular-disrupting activity of BPR0L075 in human breast cancer mammary fat pad xenografts using dynamic bioluminescence imaging. A single dose of BPR0L075 (50 mg/kg, intraperitoneally (i.p.)) induced rapid, temporary tumor vascular shutdown (at 2, 4, and 6 hours); evidenced by rapid and reproducible decrease of light emission from luciferase-expressing orthotopic MCF7 and MDA-MB-231 breast tumors after administration of luciferin substrate. A time-dependent reduction of tumor perfusion after BPR0L075 treatment was confirmed by immunohistological staining of the perfusion marker Hoechst 33342 and tumor vasculature marker CD31. The vasculature showed distinct recovery within 24 hours post therapy. A single i.p. injection of 50 mg/kg of BPR0L075 initially produced plasma concentrations in the micromolar range within 6 hours, but subsequent drug distribution and elimination caused BPR0L075 plasma levels to drop rapidly into the nanomolar range within 24 h. Tests with human umbilical vein endothelial (HUVEC) cells and tumor cells in culture showed that BPR0L075 was cytotoxic to both tumor cells and proliferating endothelial cells, and disrupted pre-established vessels <em>in vitro</em> and <em>ex vivo</em>. In conclusion, BPR0L075 caused rapid, albeit, temporary tumor vascular shutdown and led to reduction of tumor perfusion in orthotopic human breast cancer xenografts, suggesting that this antimitotic agent may be useful as a vascular-disrupting cancer therapy.</p> </div

Fluorescence images of mammary breast tumor response to administration of BPR0L075 at successive time points.

<p>Sequential images of a single nude mouse with orthotopic MCF7-<i>lacZ</i> and MCF7-luc-GFP-mCherry breast tumors growing in the front right (blue circle) and left mammary fat pad, respectively. Fluorescent signal (mCherry) increased over 24 hours following administration of carrier vehicle (A), but signals diminished 4, 6, and 24 hours after injection of BPR0L075 (B); (C) Variation in normalized fluorescent signal intensity for the group of three MCF7-luc-GFP-mCherry tumors in response to vehicle injection; (D) Variation in normalized fluorescent signal intensity for the group of six MCF7-luc-GFP-mCherry tumors in response to injection of BPR0L075. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.0001; ⁺ only three tumors observed at this time point.</p