1. Volume 7, Issue 2, Pages 174-184 (February 2003)
Systemic administration of a recombinant adenovirus encoding a HSA–Angiostatin kringle 1–3 conjugate inhibits MDA-MB-231 tumor growth and metastasis in a transgenic model of spontaneous eye cancer 
C Bouquet, E Frau, P Opolon, E Connault, M Abitbol, F Griscelli, P Yeh, M Perricaudet 
Molecular Therapy 
Volume 7, Issue 2, Pages (February 2003)
DOI: /S (02)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Construction and characterization of AdK3-HSA adenovirus. (A) AdK3-HSA, AdK3, and AdCO1 are E1/E3-deleted adenoviruses. The expression cassettes of human angiostatin (K3) and angiostatin–HSA (K3–HSA) are shown under the Ad5 genome. The human plasminogen secretion signal is represented by a blackened box. (B) Analysis of protein secretion by Western blotting from MDA-MB-231 uninfected cells (lane 1) or those infected at an m.o.i. of 300 pfu/cell with AdCO1 (lane 2), Ad K3 (lane 3), or AdK3-HSA (lane 4) adenoviruses. 30 μg of total protein was applied to each lane. The bands corresponding to angiostatin (38 kDa) and angiostatin–HSA (107 kDa) are indicated with arrows.
Molecular Therapy 2003 7, DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Inhibition of endothelial cell proliferation. HMEC-1 cells were infected at different m.o.i. for 96 h with AdK3-HSA, AdK3, and Ad CO1.
Molecular Therapy 2003 7, DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Expression of K3 and K3–HSA in nude mice sera. Sera were collected at different times after a single systemic injection of 5 × 109 pfu of AdK3 or AdK3-HSA adenovirus. Quantification of the K3 (A) and K3–HSA (B) circulating levels was performed by ELISA using a K1–3 standard (see Materials and Methods). Each triangle represents individual mouse data, and the bar graph depicts the mean of data (n = 9).
Molecular Therapy 2003 7, DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Systemic treatment of MDA-MB-231 mammary tumors by AdK3-HSA. MDA-MB-231 carcinoma cells were sc implanted into athymic nude mice. 5 × 109 pfu of adenovirus AdK3-HSA (n = 12), AdK3 (n = 10), or AdCO1 (n = 8) was iv injected when the tumors had reached a mean volume of 30 ± 8 mm3 (day 0). Data represent the tumor volume (mean ± SD) for each group. A Student t test was performed for statistical analysis (P < 0.005).
Molecular Therapy 2003 7, DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Immunohistological analysis of the MDA-MB-231 tumor vascularization after systemic treatment. 5 × 109 pfu of each adenovirus was iv injected into nude mice bearing 30 ± 8-mm3 tumors. (A) Paraffin-embedded MDA-MB-231 sections from AdCO1-, Ad K3-, or Ad K3-HSA-injected mice were submitted to α-smooth muscle actin immunostaining 28 days p.i. (original magnification ×100). (B) Vascularization scores of treated tumors. Each tumor slide was digitized, and the actin-positive vessels (immunostained in brown) were quantified by image analysis, which provides a mean score for each sample. Typical vessels are indicated with arrows. The AdK3 and AdK3-HSA scores were significantly different from that of the AdCO1 group (Student t test, P = and P = , respectively).
Molecular Therapy 2003 7, DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
AdK3-HSA delays growth of MDA-MB-231 tumors. 5 × 109 pfu of adenovirus AdK3-HSA (•, n = 6), AdK3 (▴, n = 6), or AdCO1 (•, n = 6) were iv injected into nude mice 24 h before sc injection of MDA-MB-231 cells. Mean volume values are represented with their standard deviation. Statistical significance between the AdK3-HSA and the AdCO1 groups was determined with a Student t test (P < 0.05 at day 32).
Molecular Therapy 2003 7, DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Inhibition of metastasis in TRP-1 transgenic mice. Newborn TRP-1 male mice were iv injected at days 3 and 15 after birth with 109 pfu of AdCO1 (n = 16), AdK3 (n = 6), and AdK3-HSA (n = 13) viruses. (A) Metastases incidence. A χ2 test was performed for statistical analysis (P < 0.001). (B) Volume of metastases in mice that developed brain nodules (mean volume ± SD). Statistical significance was determined with a Student t test (P < 0.035). (C) Histological analysis of these metastases after HES coloration (original magnification ×25). The metastases are indicated by arrows.
Molecular Therapy 2003 7, DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

9. FIG. 8
Expression levels of K3–HSA in the transgenic TRP-1 mice. Sera were collected at different times after the two iv injections of 109 pfu of AdK3-HSA (n = 13) and analyzed by ELISA (see Materials and Methods). Each triangle represents individual mouse data, and the bar graph depicts the mean of data.
Molecular Therapy 2003 7, DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

10. FIG. 9
Survival of male TRP-1 mice treated with AdK3-HSA. Male transgenic mice were iv injected at 3 and 15 days of age with 109 pfu of AdCO1 (n = 14) or AdK3-HSA (n = 13). Statistical significance was determined using a Kaplan–Meier test (P < 0.001).
Molecular Therapy 2003 7, DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions