Molecular Therapy - Nucleic Acids E-selectin Targeting PEGylated-thioaptamer Prevents Breast Cancer Metastases  Yoshihiro Morita, Mohamed Kamal, Shin-Ae Kang, Roy Zhang, Ganesh LR Lokesh, Varatharasa Thiviyanathan, Nafis Hasan, Sukyung Woo, Daniel Zhao, Macall Leslie, Stephen Suh, Wajeeha Razaq, Hallgeir Rui, David G Gorenstein, David E Volk, Takemi Tanaka  Molecular Therapy - Nucleic Acids  Volume 5, (January 2016) DOI: 10.1038/mtna.2016.103 Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Structure of truncated ESTAs. Truncated ESTA was designed to span either one or two stem loops. ESTA5 contains stem loop-1, ESTA6 contains stem loop-2, ESTA7 contains stem loop-1 and -2, and ESTA9 contains loop- 2 and -3. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.103) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 ESTA7 inhibits the interaction of E-selectin/CD44. Each truncated ESTA (100 nmol/l) was incubated with recombinant human E-selectin-Fc coupled IgG magnetic beads or IgG magnetic beads alone, and then incubated with 100 µg of cell lysate isolated from CD44high MDA-MB-231. The interaction of E-selectin and CD44 was analyzed by western blot using anti-pan-CD44 and anti-E-selectin antibodies. Band intensity was measured by densitometry and the degree of inhibition for E-selectin/CD44 interaction achieved by full-length ESTA was defined as 100% and by control TA with a random sequence as 0%. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.103) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 ESTA7 inhibits shear-resistant adhesion of CD44high BCa. Shear-resistant adhesion of BCa to ESTA-pretreated ES-HMVECs was evaluated. The cells were incubated with doxycycline or saline for 4 hours for the induction of E-selectin expression. (a) ES-HMVECs were treated with ESTAs (100 nmol/l) or saline for 1 hour at 37 °C. A single suspension of MDA-MB-231 or MDA-MB-468 cells at 5 × 105 cells/ml was infused into a flow chamber at 1 dyne/cm2. Shear-resistant adhesion of BCa to doxycycline-treated/TA-incubated ES-HMVECs was compared to doxycycline-treated/saline-incubated one (defined as 100%). (b) Doxycycline-treated ES-HMVECs were further treated with various concentrations of ESTA7 or ESTA (0, 0.001, 0.01, 0.1, 1, 10, and 100 nmol/l) for 1 hour at 37 °C. The adhesion of MDA-MB-231 cells to doxycycline-treated/saline-incubated ES-HMVECSs was defined as 100%. (c) Doxycycline-treated ES-HMVECs were further treated with saline, ESTA7, or ESTA for 1 hour. The MDA-MB-231 cells were then infused into the chamber for 5 minutes under different shear stress (1.0, 1.5, and 2.0 dyne/cm2). The shear-resistant adhesion of BCa to doxycycline-treated ES-HMVEC at 1.0 dyne was defined as 100%. The data represent mean ± SD from at least duplicate of three independent experiments, and statistical significance was determined by nonparametric Student's t-test. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.103) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 PEGylation of ESTA7 does not hamper metastasis inhibition. (a) The secondary structure of PEGylated ESTA7. (b) A monolayer of ES-HMVECs was incubated with saline or doxycycline, followed by ESTA, ESTA7, or control TA conjugated with various lengths of PEG at sizes of 2, 5, 10, and 20 kDa (100 nmol/l) for 1 hour at 37 °C. A single suspension of MDA-MB-231 cells (5 × 105cells/ ml) was infused into a parallel flow chamber at 1 dyne/cm2. (c) Doxycycline-treated HMVECs were incubated with various concentrations of ESTA7, ESTA7-p5, or ESTA7-p10 (0, 0.001, 0.01, 0.1, 1, 10, and 100 nmol/l) for 1 hour at 37 °C. The data represent mean ± SD from triplicated experiments, and statistical significance was determined by nonparametric Student's t-test. Shear-resistant adhesion of BCa to doxycycline-treated/saline incubated-ES-HMVECs was defined as 100%. (d) Female athymic nu/nu mice (n = 5) were preconditioned with IV injection of 10 ng VEGF-A. Two hours later, ESTA, ESTA7, ESTA7-p5, ESTA7-p10, or control TA (45 µmol/l/100 µl saline) were injected via the tail vein. The mice were then injected with MDA-MB-231-TGL cells (3 × 104 cells) via the tail vein at 6-hour intervals. Total photon flux was measured and 3 representative images are shown. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.103) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 ESTA7-p10 inhibits metastasis via E-selectin. (a) Preconditioned female athymic nu/nu mice (n = 4–5) were injected via the tail vein with ESTA7-p10 at a concentration of 0.45, 4.5, or 45 µmol/l in 100 µl saline. The mice were then injected with MDA-MB-231-TGL cells (3 × 104 cells) via the tail vein at a 6-hour interval. (b) Female athymic nu/nu mice (n = 3–4) were preconditioned with IV injection of conditioned media for 3 consecutive days. Following preconditioning, the mice were injected with saline, ESTA7-p10, or control TA (45 µmol/l in 100 µl saline) once via the tail vein. The mice were then injected with MDA-MB-231-BR cells (5 × 104 cells) via the left ventricle at a 6-hour interval. (c) Female E-selectin K/O and sex- and age-matched wild-type mice (n = 3–4) were preconditioned for 4 weeks with IP injection of conditioned media from 4T1 murine BCa cells. The mice then received a single IV injection of ESTA7-p10 (45 μmol/l in 100 μl saline) or saline, followed by 4T1-Luc BCa (5 × 104 cells). Lung metastasis formation was measured by bioluminescent imaging 3–5 weeks after cancer cell inoculation. The data was compared to saline treated mice and statistical significance was determined by nonparametric Mann–Whitney test. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.103) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 ESTA7-p10 prolongs circulation half-life. Pharmacokinetic parameters of ESTA7-p10 were determined in female ICR mice. (a) Elimination of TA was measured following a single intravenous injection of Cy3-labled TA (45umol/l in 100 ul saline) via the tail vein (n = 3). Whole blood was collected by cardiac puncture at 2, 5, 10, 20, 30, 60, 120, 180, and 360 minutes after the injection. Plasma from saline-injected mice was collected as a baseline. (b) Pharmacokinetics parameters were calculated using a two-compartment model. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.103) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 ESTA7-p10 inhibits metastasis superior to ESTA in a spontaneous metastasis model of breast cancer. (a) For spontaneous metastasis, 4T1 (5 × 105) was injected into the mammary fat pad of 5-week-old female Balb/C mice (n = 5). When the tumor became palpable, the mice received intravenous injection of ESTA7-p10, ESTA, or saline every 3 days. Three weeks after surgical removal of the primary tumor (500–700 mm3), metastasis was measured by bioluminescence. (b) Enzymatic activities were measured from plasma collected 1 hr after injection of the effective dose of ESTA7-p10 (n = 5). Control mice received bolus intravenous injection of 100 µl saline. The data represent mean ± SD. Statistical significance was determined by nonparametric Student t-test. (c) C3a and C5a activities were measured from whole blood collected from mice that received either single or repeated dose of ESTA7-p10 (n = 5). Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.103) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions