Vevo 2100 ultrasound imaging system

Transthoracic echocardiography was performed using the Vevo 2100 ultrasound imaging system (VisualSonics, Toronto, Canada) after anesthetization by isoflurane inhalation on day 7, day 14 and day 28 following virus injection. The echocardiographic data such as left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were then measured blindly according to the operator’s manual.

For echocardiography, a high-resolution Vevo2100 Ultrasound imaging system (Visual Sonics Inc., Toronto, Canada) was used as described previously.²⁸ (link) Animals at the ages of 10, 12, and 16 weeks were imaged under inhalation anaesthesia [induction with 3.5–4% isoflurane (Baxter International Inc., Deerfield, IL, USA) and 350–400 mL/min air, maintenance with 2–2.5% isoflurane and 200–250 mL/min air] and kept on a heated platform during and shortly after the imaging.

The mECE20 lentivirus mediated transgenic mice were generated as previously described [24 (link),44 (link)]. Briefly, high-titer of lentiviral particles (mECE20 mixed with mCherry which allows the visualization of infected mice at harvest) were delivered into the amniotic cavity of embryonic day (E) 9 CD1/NCrl mouse embryos (Charles River Labs). Injections were performed under ultra-sound guidance using the Vevo 2100 ultrasound imaging system (Visualsonics, Toronto Canada) equipped with a 35–50 MHz mechanical transducer as described previously [24 (link),44 (link)]. Primers used for cloning are reported in S1 Table. All survival surgeries were carried out in accordance with approved IACUC protocols. See also Table 1.
P 2.5 pups were sacrificed and both forelimbs, and both hindlimbs were harvested from mCherry positive animals. Limbs were processed by cryosection and immunohistochemical analysis was performed as described to detect GFP positive clones. See also Table 1.

Tumor blood perfusion analysis was performed on day 21 (4T1 and 67NR models) or on day 19 (E0771 model) using the MicroMarker™ contrast agent (VisualSonics, Toronto, ON, Canada) and Vevo 2100 ultrasound imaging system (VisualSonics). Mice were anesthetized by continuous administration of 2–3% (v/v) isoflurane (Baxter, Deerfield, Germany) in synthetic air (600 mL/min) and immobilized. The tumor area was enclosed with air-bubble-free gel, and the central cross-section of the tumor was visualized in the transverse plane using an MS250 scanhead (VisualSonics). Then, 100 μL of the contrast agent was injected intravenously and the first imaging sequence (bolus) was recorded (ca. 15 fps, at least 1000 frames) after the contrast signal in the tumor tissue reached the steady state (ca. 50 s). Next, using the burst mode, contrast microbubbles were destroyed, and the second imaging sequence (replenishment) was recorded. Mice were maintained in a warm environment until fully awakened. The data were analyzed using the Vevo LAB 1.7.1 Software with VevoCQ modality (VisualSonics).

A Vevo2100 Ultrasound imaging system (VisualSonics Inc., Toronto, ON, Canada) was used for in vivo cardiac imaging as previously [6 (link)]. Mice were imaged under inhalation anaesthesia induced with 4% isoflurane (Baxter International Inc., Deerfield, IL, USA) and 400 mL/min air. Anaesthesia was maintained with 2% isoflurane and 200 mL/min air. During and shortly after imaging mice were kept on a heated platform. Cardiac parameters were assessed from short-axis M-Mode measurements.