Journal: Nmr in Biomedicine

Article Title: Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using α v β 3 ‐targeted iron oxide microparticles

doi: 10.1002/nbm.4948

Figure Lengend Snippet: (A) Representative MR images from mice bearing 4T1‐GFP macrometastases at Days 28–35. (Ai,ii) Tumours imaged with RGD‐MPIO; (Ai) Nonenhancing tumour at Day 28, (Aii) Gadolinium‐enhancing tumour at Day 35. (Aiii–iv) Tumours imaged with RDG‐MPIO; (Aiii) Nonenhancing tumour at Day 28, (Aiv) Gadolinium‐enhancing tumour at Day 35. Each column of images, from the left, shows T 1 ‐weighted postgadolinium images, T 2 *‐weighted pre‐MPIO MGE3D images, T 2 *‐weighted post‐MPIO MGE3D images, and overlays on T 2 *‐weighted MGE3D images showing hypointensities pre‐MPIO and post‐MPIO. For the overlays, the tumour‐bearing striatum is segmented in green, and the contralateral striatum segmented in pink. Hypointense voxels are shown in red. (B) Significantly increased RGD‐MPIO–induced hypointense voxels were evident in the tumour‐bearing striatum (white bars) compared with the contralateral striatum (black bars) at Day 35 (two‐way paired ANOVA, p < 0.05). (C) Significantly increased control RDG‐MPIO–induced hypointense voxels were also seen in the tumour‐bearing striatum (white bars) compared with the contralateral striatum (black bars) at Day 35 (two‐way paired ANOVA, p < 0.05). (D–E) Comparison of pooled data across all timepoints for (D) Nonenhancing tumours, and (E) Gadolinium‐enhancing tumours. (D) Mice with nonenhancing tumours administered RGD‐MPIO (white bars; n = 7) showed significantly increased MPIO‐induced hypointense voxels in the tumour‐bearing hemisphere (one‐way ANOVA, p < 0.005) than both the contralateral hemisphere and the mice administered control RDG‐MPIO (black bars; n = 13) in the tumour‐bearing hemisphere. (E) In mice with gadolinium‐enhancing tumours, significantly increased MPIO‐induced hypointense voxels were observed in the tumour‐bearing striatum compared with the contralateral striatum for both RGD‐MPIO (white bars; n = 10) and RDG‐MPIO (black bars; n = 3) (one‐way ANOVA, p < 0.0001). However, in mice receiving control RDG‐MPIO, the volume of MPIO‐induced hypointense voxels was also significantly greater than those administered RGD‐MPIO. Number of MPIO‐induced hypointense voxels, presented as postcontrast minus precontrast hypointense voxels for all data. Bars represent mean ± standard deviation; post‐hoc Holm–Sidak's tests. * p < 0.05, *** p < 0.001. MGE3D, multigradient echo three‐dimensional; MPIO, microparticles of iron oxide; RDG, Arg‐Asp‐Gly peptide, scrambled control; RGD, Arg‐Gly‐Asp peptide, targeting integrin α v β 3 .

Article Snippet: Subsequently, capillaries were filled with 200 ng/mL mouse integrin α v β 3 (7889‐AV‐050, R&D Systems, Abingdon, UK) and incubated at room temperature for 24 h. The remaining functional groups were quenched with 10 mM ethanolamine for 24 h at room temperature.

Techniques: Control, Comparison, Standard Deviation

Journal: Nmr in Biomedicine

Article Title: Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using α v β 3 ‐targeted iron oxide microparticles

doi: 10.1002/nbm.4948

Figure Lengend Snippet: Aggregations of iron in 4T1‐GFP tumour tissue. (A) Representative section of gadolinium‐enhancing tumour stained for Perls' Prussian blue (iron, blue) and counterstained with nuclear fast red. Black arrowheads indicate examples of single MPIO associated with the vascular endothelium, and red arrowheads indicate examples of iron aggregations. (B–C) Correlations between iron aggregations and tumour size in mice injected with either (B) RGD‐MPIO or (C) Control RDG‐MPIO. Linear regression analysis showed a positive correlation between number of aggregations and tumour size ( R 2 = 0.64, *** p < 0.001) in mice injected with RGD‐MPIO, but not control RDG‐MPIO, although a similar trend was evident; 95% confidence intervals are shown. (D–F) Consecutive sections stained for (D) Iba‐1 (macrophages and microglia, brown staining), (E) Prussian blue (iron), and (F) CD31 (blood vessels, brown staining) in a Day 35 mouse injected with RGD‐MPIO. The red arrow indicates a larger iron aggregation in a similar location to macrophage staining (D; Iba‐1), while the black arrow indicates single MPIO distant from macrophage staining, but close alignment with a blood vessel (F; CD31). (G–H) Double staining of 4T1‐GFP tumour tissue sections, from a mouse injected with RGD‐MPIO at the Day 35 timepoint, for Prussian blue (iron) and macrophages/microglia (Iba‐1, brown staining), indicate colocalisation of iron within macrophages/microglia (red arrows). Sections counterstained with nuclear fast red. (I) Double staining for Prussian Blue and CD31 revealed single MPIO (black arrow) bound to blood vessels (brown stained) in mice injected with RGD‐MPIO; representative image from Day 21 shown. (J) In the gadolinium‐enhancing 4T1‐GFP tumours, single endothelium‐bound MPIO are observed more often in mice injected with RGD‐MPIO ( n = 10) than control RDG‐MPIO ( n = 3, t ‐test, ** p < 0.01). (K) Histogram showing the cumulative frequency of the measured distance between the centre of the iron‐laden macrophages and the centre of the blood vessel lumen, indicating their close association with blood vessels. Scale bar = 25 μm in (A) and 10 μm in (D–I). MPIO, microparticles of iron oxide; RDG, Arg‐Asp‐Gly peptide, scrambled control; RGD, Arg‐Gly‐Asp peptide, targeting integrin α v β 3 .

Article Snippet: Subsequently, capillaries were filled with 200 ng/mL mouse integrin α v β 3 (7889‐AV‐050, R&D Systems, Abingdon, UK) and incubated at room temperature for 24 h. The remaining functional groups were quenched with 10 mM ethanolamine for 24 h at room temperature.

Techniques: Staining, Injection, Control, Double Staining

Journal: PLOS ONE

Article Title: An intravenous pancreatic cancer therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi -Non Toxic

doi: 10.1371/journal.pone.0289183

Figure Lengend Snippet: (A) Pictural description of the α V β 3 adhesion assay methodology. (B) Fold change observed in crystal violet (CV) absorbance at a wavelength of 590 for wild type (WT), non-toxic (NT) C . novyi as well as the putative RGD-modified candidates (A and B) after exposure to the α V β 3 coated surface of the adhesion assay. * denotes a p value of < 0.05 when compared to any other cohort, including WT, NT, and modification Candidate B. (C) Average CV pixel count of entire integrin coated surface for candidates A and B as well as wild-type (WT) and non-toxic (NT) C . novyi that remain on the α V β 3 coated surface. ‡ denotes a p value of 0.05 when compared to any other cohort, including WT, NT, and modification Candidate B. Error bars represent standard deviation from the cumulative mean of three experimental replications (n = 6 each) for a total n = 18.

Article Snippet: Purified α V β 3 integrin (100μL of 10μg/mL carrier-free, human recombinant protein, R&D Systems Bio-Techne 3050-AV) solution was administered to the center of the corona-treated cover slips.

Techniques: Cell Adhesion Assay, Modification, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Analysis of in situ and ex vivo αVß3 integrin expression during experimental carotid atherogenesis

doi: 10.2147/ijn.s28065

Figure Lengend Snippet: Figure 4 Accumulation of macrophages in the stenotic segment. (A) Representative micrograph of carotid artery sections stained with antibodies against αvβ3 integrin (400× magnification, scale bar = 100 µm). (B) Representative images of MAC-3 immunostaining in the stenotic lesion areas of the left carotid artery (400× magnification, scale bar = 100 µm). (C) Quantitative analyses of MAC-3-positive cells. (D) Colocalization in the proximal neointima, blue fluorescence of DAPI, the red fluorescence of Cy5.5 (probe) and the green fluorescence of FITC (macrophage) were examined (800× magnification). Notes: Upper panel shows representative photomicrographs of cross sections of left carotid artery proximal carotid site, white arrows show positive staining. Lower panel shows representative photomicrographs of cross sections of right carotid artery. Abbreviation: DAPI, diamidino-2-phenylindole dihydrochloride.

Article Snippet: Primary antibody against α V β 3 integrin (anti-human integrin α V β 3 CD51/CD61, R&D Systems; Minneapolis, MN, 1:200) was used for immunostaining of α V β 3 integrin.

Techniques: Staining, Immunostaining, Fluorescence

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Self-Triggered Apoptosis Enzyme Prodrug Therapy (STAEPT): Enhancing Targeted Therapies via Recurrent Bystander Killing Effect by Exploiting Caspase-Cleavable Linker.

doi: 10.1002/advs.201800368

Figure Lengend Snippet: Figure 2. Enhanced cellular uptake and cytotoxic activity of RGDEVD-DOX in integrin αvβ3 expressing cells. a) Representative confocal images (left) and quantitative analysis (right) of HDMEC and U-87 MG cells exposed to fluorescent-labeled RDEVD and RGDEVD peptides. b) Representative confocal images (left) and quantitative analysis (right) of scrambled control and integrin αv (ITGAV) siRNA-transfected HDMECs and U-87 MG cells exposed to fluorescent-labeled RGDEVD peptide. Green and blue indicate the fluorescent-labeled peptides and cell nuclei, respectively. Scale bar, 50 µm. c) Flow cytometry analysis of isotype control (top), scrambled control-transfected (lower left), and ITGAV siRNA-transfected (lower right) U87 MG cells incubated with fluorescent-labeled RGDEVD and stained with an antibody against integrin αvβ3. d) Representative confocal images (left) and quantitative analysis (right) of U-87 MG and HT-29 cells treated with RDEVD-DOX and RGDEVD-DOX. Red and blue indicate the intrinsic red fluorescence of doxorubicin and cell nuclei, respectively. Scale bar, 50 µm. e) Concentration-dependent cytotoxicity of RDEVD-DOX and RGDEVD-DOX on U-87 MG (left) and HT-29 (right) determined by MTT assay (n = 4). f) Doxorubicin release from RGDEVD-DOX when incubated in PBS (pH 7.4) containing (or not containing) carboxylesterase (n = 3). Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The cells were incubated with Alexa Fluor 488-conjugated integrin αvβ3 antibody (1:100; R&D Systems, Minneapolis, MN; Cat. No. FAB3050G) for an hour at 4 °C, washed, and suspended in PBS containing 0.5% BSA.

Techniques: Activity Assay, Expressing, Labeling, Control, Transfection, Flow Cytometry, Incubation, Staining, Fluorescence, Concentration Assay, MTT Assay