B6.3 diabody inhibited LAP-mediated Smad2/3 translocation to the nucleus in Capan-1 cells.

<p>Cells were incubated at 4°C in the presence of B6.3 diabody and then treated with LAP or TGFβ₁ (30 min, 37°C); Smad2/3 localization was assessed by confocal microscopy (40X) using rabbit anti-Smad2/3 followed by Alexa Fluor 488®-labeled goat anti-rabbit IgG (green); smad2/3 was found in the cytoplasm of starved Capan-1 cells (a) and after treatment with B6.3 diabody (c). Smad2/3 was present in the nuclei in response to treatment with latent TGFβ₁ (b), a translocation that was inhibited by pre-treatment B6.3 diabody (d). TGFβ₁ was used as a positive control (e.f).</p

Treatment of α_vβ₆-expressing cells with B6.3 diabody resulted in diabody internalization and blockade of integrin functions.

<p>A) Localization of B6.3 diabody in A375Pβ6 cells by confocal microscopy. B6.3 diabody detection showed membrane pattern of staining at 4°C and internalized when cells were incubated at 37°C for 30 min, 1 h and 3 h. B6.3 diabody was detected using rabbit anti-human IgG followed by Alexa Fluor 546®-labeled goat anti-rabbit IgG (red). Cells were also counterstained with Hoechst 33245 (blue). B) Treatment of α_vβ₆-expressing cells blocked adhesion to LAP-coated plates (A375Pβ6 and Capan-1 cells) and/or fibronectin-coated plates (Capan-1 cells). Cells were incubated with B6.3 or shMFE23 diabody at 4°C for 1 h and allowed to attach to coated plates for 1 h at 37°C. Treatment with the anti-CEA shMFE23 diabody had no effect on the cell lines used. C) B6.3 diabody treatment inhibited migration towards LAP and fibronectin. As observed in adhesion assays, the diabody inhibited migration of A375Pβ6 cells to LAP and migration of Capan-1 cells to fibronectin and LAP, while targeting CEA had no effect on the cells tested.</p

Labeling with ^99mTc did not affect B6.3 diabody binding to α_vβ₆.

<p>A) Saturation Binding experiment showed concentration-dependent binding of ^99mTc-labeled diabody to A375Pβ6 cells. Non-specific binding, including 25 µg of unlabeled diabody was subtracted from each data point. KD obtained was 4.88±0.32×10⁻⁸ M and BMax was 2.3±0.039×10⁵ receptors/cell (325±5.53 pM/8.5×10⁵ cells). B) Scatchard presentation of the data. Each experiment was carried out in duplicate.</p

^99mTc-labeled B6.3 diabody localised specifically to α_vβ₆-expressing tumors in vivo.

<p>A) A375Pβ6 and A375Ppuro cells were injected subcutaneously on opposite shoulders and ^99mTc-labeled B6.3 diabody (approximately 11 µg, 30 MBq) was injected intravenously once tumours had developed. Mice were imaged by SPECT/CT as indicated 2 h, 5 h and 24 h after injection. B) SPECT/CT cross sections of the same mice at 2, 5 and 24 h. C) Percent injected doses of ^99mTc-labeled B6.3 diabody in A375Pβ6 and A375Ppuro tumours from three mice, obtained from these images. D) Biodistribution of ^99mTc-labeled B6.3 diabody 24 h after injection. Data expressed as % injected dose/g (%ID/g) as mean ± SD for 5 animals. Tumor-to blood ratios at this time point were 40.4 for A375Pβ6 tumors and 15.5 for A375Ppuro tumors. Significance assessed by Student's t-test.</p

Production of B6.3 diabody and analysis of its specific interaction with α_vβ₆.

<p>A) Size-exclusion chromatographic profile (Superdex 75, 125 ml) of B6.3 diabody after fermentation, expanded-bed adsorption IMAC, Superdex 75 (500 ml), 1 ml Ni²⁺-charged Hi-Trap IMAC, freezing and de-frosting. B6.3 diabody eluted from the column as a dimer that separated in monomeric form under reducing conditions by SDS-PAGE, consistent with non-covalent association of monomers in a diabody structure. B) Sensogram of real-time binding and dissociation of B6.3 diabody to α_vβ₆. B6.3 diabody was immobilized on a BIAcore CM5 sensor chip and α_vβ₆ protein was flown across at 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 nM. The affinity constant (KD) for the interaction was 2.8×10⁻⁹ M, with on-rate of 8,107±7.3 M⁻¹s⁻¹ and off-rate of 2.3×10⁻⁵±1.4×10⁻⁷ s⁻¹. C) Flow cytometry analysis of B6.3 diabody binding to α_vβ₆-expressing A375Pβ6 cells in a concentration-dependent manner (a) but not to A375Ppuro cells (b), which do not express this integrin. Cells were incubated with B6.3 diabody, at the indicated concentrations and binding was detected with mouse anti-tetra-histidine IgG followed by R-PE-labeled goat anti-mouse IgG. B6.3 diabody was not added to omission control (shown in solid grey). D) Inhibition of B6.3 diabody binding after incubation with the anti-α_vβ₆ antibody 10D5 shown by flow cytometry. Cells were incubated with 100 ng B6.3 diabody with or without prior incubation with 10D5 at the indicated concentrations. Binding of B6.3 diabody was detected with rabbit anti-hexahistidine IgG followed by R-PE-labeled goat anti-rabbit IgG. In the omission control experiment (shown in solid grey) cells were not incubated with B6.3 diabody and 10D5.</p

Genetic ablation of Rac1 in endothelial cells does not impair tumor growth or tumor angiogenesis.

<p><b>A.</b> Western blot analysis of Rac1 flox/flox PDGFB-iCreER OHT-treated primary endothelial cell extracts showed that Rac1 was barely detectable (lane 3) when compared with Rac1 flox/flox OHT-treated (lane 1) or Rac1 flox/flox PDGFB-iCreER vehicle treated (lane 2) endothelial cell extracts (left panel). Western blots of extracts from non-endothelial cells, treated as described above, showed no differences in Rac1 expression (right panel). Hsc-70 provided loading controls. Bar graphs represent densitometric values relative to Hsc-70. N = 2 independent experiments. <b>B.</b> B16F0 tumor volume and angiogenesis (blood vessel density) from tumors grown in OHT-treated Rac1 flox/flox (1, white), or placebo- (2, grey) or OHT- (3, black) treated Rac1 flox/flox PDGFB-iCreER mice. Left bar graph shows mean tumor volume in mm³ (+ s.e.m) for 10-day-old tumors. No significant differences in tumor size were observed between groups. N = 6–7 animals per group. Right bar graph shows mean number of PECAM-1 positive vessels per tumor area per mm² (+ s.e.m.) for 10-day-old tumors. No significant differences in microvessel density were observed between groups. N = 4 animals per group. <b>C.</b> B16F0 tumor volume and angiogenesis (blood vessel density) from tumors grown in OHT-treated Rac1 flox/flox PDGFB-iCreER animals. Tumors were injected at days 5 and 10 (after initial inoculation) with pSico-Con (1, white) or pSico- β3 (2, black) and harvested at day 14. Left bar graph shows mean tumor volume in mm³ (+ s.e.m). <i>P</i><0.05. N = 5 animals per group. Right bar graph shows mean number of endomucin positive vessels per tumor area per mm² (+ s.e.m.). <i>P</i><0.001. N = 5 animals per group.</p

VEGF-mediated angiogenesis is dependent on endothelial-Rac1 expression in β3-null mice but not wild-type mice.

<p><b>A.</b> Representative images of 14-day-old VEGF-impregnated sponges in wild-type/Tie1-Cre⁺ and β3-null/Tie1-Cre⁺ mice after treatment with pSico-Con, pSico-Rac1 and pSico-Flk-1. Endomucin-positive staining (blue) identified microvessels. Scale bar: 50 µm. Bar graph represents mean number of microvessels per area of sponge (+ s.e.m.). Blood vessel density was reduced significantly in pSico-Flk-1-treated mice of both genotypes (*P<0.05), and in pSico-Rac1-treated β3-null/Tie1-Cre⁺ mice, but not pSico-Rac1-treated wild-type/Tie1-Cre⁺ mice (<i>n.s.d</i>, no significant differences). N = 6 sponges per group. <b>B.</b> Subcutaneous sponges implanted into wild-type mice were injected with either PBS or VEGF in the presence (Rac1 siRNA) or absence (Con siRNA) of Rac1-specific siRNA. Bar graph shows mean number of laminin-positive vessels per area of sponge per mm² (+ s.e.m). Blood vessel density was increased significantly in VEGF alone-treated (*P<0.01) when compared with PBS controls. Rac1 siRNA-treatment had no significant effect (<i>n.s.d</i>, no significant differences). N = 6–10 sponges per group. <b>C.</b> Semi-quantitative RT-PCR (left) and Western blot (right) analyzes from wild-type and β3-null aortic explants transfected with either Con or Rac1 specific-siRNA. Rac1 siRNA reduced significantly the expression of both Rac1 mRNA (*P<0.01) and protein (*P<0.001) levels. RT-PCR for Αctin and Western blotting for Hsc-70 provided mRNA and protein loading controls, respectively. Bar graphs represent mean Rac1 mRNA and Rac1 protein levels (+ s.e.m.). N = 3 independent experiments. <b>D. </b><i>Ex vivo</i> mouse aortic ring assays. Representative high-power light micrographs of VEGF-mediated microvessel sprouting from wild-type and β3-null mouse aortic rings treated with Con or Rac1-specific siRNA and Con siRNA plus DC101. Bar graphs represent quantification of microvessel numbers from 5-day-old VEGF-stimulated aortic ring cultures transfected with Mock, Con or Rac1 siRNA in the presence or absence of DC101. Sprouting angiogenesis was reduced significantly after DC101 treatment of aortas and in Rac1-specific siRNA transfected β3-null, but not wild-type, aortas. Bar graph represents mean number microvessel sprouts/aortic ring (+ s.e.m.).*P<0.01; <i>n.s.d</i>, no significant differences. N = 3-5 independent experiments.</p

Endothelial-specific Rac1-depletion does not impair tumor growth wild-type mice but does in β3-null mice.

<p><b>A.</b> Murine B16F0 cells (10⁶) were injected subcutaneously into the flanks of wild-type/Tie1-Cre⁺ or β3-null/Tie1-Cre⁺ mice. Lentiviral vector suspensions (10⁶ i.u/ml) of pSico-Con, pSico-Rac1 and pSico-Flk-1 were injected intratumorally on days 5 and 10 after tumor cell injection. Representative macroscopic appearance of 14-day-old B16F0 pSico-Con-, pSico-Rac1- and pSico-Flk-1- treated melanomas in both genotypes. Scale bar: 5 mm. Bar graph shows mean tumor volume per mm³ (+ s.e.m.). Tumor size was reduced significantly in pSico-Flk-1-treated mice of both genotypes (*P<0.05) and in pSico-Rac1 treated β3-null/Tie1-Cre⁺ but not in pSico-Rac1-treated wild-type/Tie1-Cre⁺ mice (<i>n.s.d</i>, no significant differences). N = 4–6 animals per condition. <b>B.</b> Representative merged images of PECAM-1 (red) and GFP (green) -immunostained sections from pSico-Rac1-treated B16F0 tumors grown in wild-type, wild-type/Tie1-Cre⁺, β3-null and β3-null/Tie1-Cre⁺ mice. PECAM-1-positive staining identified endothelium. GFP-positive staining was observed in tumor cells (concave arrowheads) and in PECAM+ endothelium (arrows) of blood vessels in B16F0 tumors from wild-type and β3-null control mice, indicating successful pSico-Rac1 lentivirus infection <i>in vivo</i>. Loss of GFP detection in most PECAM-1-positive microvessels (small arrowheads), but not in B16F0 tumor cells, was observed in pSico-treated tumors grown in wild-type/Tie1-Cre⁺ and β3-null/Tie1-Cre⁺ mice, indicating successful endothelial-specific Cre recombination <i>in vivo</i>. Scale bar: 10 µm. Bar graph shows mean numbers of PECAM-1⁺/GFP⁻ vessels per unit area of tumor section per mm² (+ s.e.m). Blood vessel density was reduced significantly in pSico-Flk-1-treated mice of both wild-type/Tie1-Cre⁺ and β3-null/Tie1-Cre⁺ mice (*P<0.05) and in pSico-Rac1-treated β3-null/Tie1-Cre⁺ mice but not pSico-Rac1-treated wild-type/Tie1-Cre⁺ mice (<i>n.s.d</i>, no significant differences). N = 4 animals per condition.</p

Increased active levels of Rac1 in β3-integrin-deficient endothelial cells.

<p><b>A.</b> Active levels of Rac1 were examined by GST-PAK pull-downs. Western blot analysis of active Rac1 bound to GST-PAK (Rac1-GTP) and total Rac1 from wild-type, β3-integrin null, and β3-null primary lung endothelial cells transduced with human β3-integrin (rescue). Immunoblots were quantified by densitometry, and the levels of GTP-bound Rac1 normalised to total Rac1 levels. Active levels of Rac1 were increased approximately 3 fold in β3-null endothelial cells when compared with wild-type controls (*P<0.01), however, total levels of Rac1 were expressed equally in both genotypes. Furthermore, active levels of Rac1 were reduced to wild-type levels in rescue cells (*P<0.01). Results shown are the means + s.e.m of 3-4 independent experiments. <b>B.</b> Flow-cytometric analysis shows that surface levels of β3-integrin were not detectable in β3-null (dashed line) cells when compared with wild-types (bold line) endothelial cells (left panel). Rescue cells expressed β3-integrin (bold line, right panel). Grey peaks represent isotope IgG controls. Bar graph shows means + s.e.m of relative surface β3-integrin expression in wild-type (white), β3-null (black) and rescue (grey) endothelial cells compared with negative control (*P<0.001, N = 3 independent experiments).</p

Cre-regulated depletion of Rac1.

<p><b>A.</b> Wild-type primary endothelial cells were infected with pSico-Rac1 lentivirus. High-efficiency transduction was achieved as indicated by uniform GFP expression in infected cells (left panel). Cells sorted for GFP positivity and transfected with a Cre-recombinase expression plasmid (pTURBO-Cre) showed a significant loss of GFP (right panel). Scale bar: 10 µm. <b>B.</b> One week after pTURBO-Cre transfection, levels of mRNA, detected by semi-quantitative RT-PCR (upper panels), and protein, detected by Western blotting (lower panels), showed successful Rac1- and Flk-1-depletion in pSico-Rac1 and pSico-Flk-1 infected cells, respectively. Αctin RT-PCR and Western blotting for Hsc-70 were carried out to ensure equal RNA and protein loading, respectively. Bar graphs represent densitometric readouts of mRNA of relative Actin or Hsc-70 protein levels, respectively. *P<0.01. N = 3 independent experiments.</p