Intratumoral injection technique, imaging acquisition and data analysis

Intratumoral injections were performed in a hybrid image-guided intervention suite. Injection needle placement was performed by an interventional radiologist under real-time ultrasound guidance (Acuson, Siemens). Ultrasound imaging was used to confirm that the needle tip was located within the center of the target lesion. Injection of iodinated contrast agents and/or immunotherapeutic agents was performed under live fluoroscopic imaging at 15 frames-per-second (Artis Q, Siemens). The animals were then scanned using a CT-on-rails (Miyabi, Siemens) immediately following injection; this allowed the animals to remain stationary without having to be transferred to another imaging suite or gantry. CT slice thickness in the z-axis was 0.6 mm. Volumetric analysis of the CT images was performed to quantify the percentage of tumor filled with injected drug using conventional three-dimensional (3D) imaging analysis software (iNtuition; TeraRecon, Foster City, California, USA).

The influence of injection needle design on injection drug delivery and retention was performed in the following manner. Injections were performed using either a conventional 21 gage end-hole needle (EHN; Becton Dickinson) or a multiside hole needle with no end hole (ProFusion, Cook Regentec). The ProFusion needle used in this study has a closed diamond tip to facilitate image-guided positioning within the target lesion. The distal 1 cm of the needle has 22 laser-etched holes distributed in a spiral pattern that has a characteristic echogenic pattern. The multiple parallel channels allow improved intratumoral distribution and go hand-in-hand with diminished pressure during injection. Needle placement was performed under ultrasound guidance to ensure that all of the side holes were positioned within the tumor, for a proximal side hole positioned outside of the tumor would lead to off-target leakage.

The needles were connected to syringes, and injections of an iodinated contrast agent (Omnipaque 240) were performed simultaneously into bilateral flank tumors in the rat tumor model (n=10). Injections were performed at an equal volume (1 cc) and at a constant injection rate using a dual head injection pump (Harvard Apparatus). Continuous, in-line pressure measurements were obtained during injections using a piezoelectric pressure sensor (CompassCT, Cook Regentec). Ultrasound imaging was used intermittently to ensure that the needle remained well positioned within the target lesion.

The influence of tumor stroma composition on injection drug delivery was performed in the following manner. Intratumoral injections of iodinated contrast (Omnipaque 240) were performed in the B16 melanoma and MC38 colorectal cancer mouse models described previously. Prior to injection, tumor volumes were measured with calipers using the formula length×width²/2. The intratumoral injectate volume for each tumor was drawn up to be equal to the tumor volume calculated using this technique. Injections were performed under ultrasound guidance through a 27 gage single EHN; unlike the larger rat tumors that can accommodate a clinically relevant 21G needle, a higher needle gage was necessary for injections into the smaller mouse tumors. Animals were then immediately imaged using a microCT system (Skyscan, Bruker). Volumetric analysis of the CT images was performed to quantify the percentage of tumor filled with injected drug using a semi-automated threshold-based volumetry algorithm with a conventional 3D imaging analysis software package (iNtuition; TeraRecon).