DCE-MRI Detects Early Vascular Response in Breast Tumor Xenografts Following Anti-DR5 Therapy

Abstract

Purpose

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) measured the early vascular changes after administration of TRA-8, bevacizumab, or TRA-8 combined with bevacizumab in breast tumor xenografts.

Procedures

Groups 1–4 of nude mice bearing human breast carcinoma were injected with phosphate-buffered saline, TRA-8, bevacizumab, and TRA-8 + bevacizumab on day 0, respectively. DCE-MRI was performed on days 0, 1, 2, and 3, and thereafter tumors were collected for terminal deoxynucleotidyl transferase-mediated dUT nick end labeling and CD31 staining.

Results

DCE-MRI measured a significant K ^trans change within 3 days after TRA-8 therapy that correlated with tumor growth arrest, which was not shown with statistical significance by histopathology at these early time points posttreatment. The K ^trans changes followed quadratic polynomial curves.

Conclusion

DCE-MRI detected significantly lower K ^trans levels in breast tumor xenografts following TRA-8 monotherapy or combined therapy with bevacizumab.

Appendices

Appendix A: Mouse Port Implantation and Maintenance

Each mouse was anesthetized with an intraperitoneal injection of sodium pentobarbital (60–65 mg/kg BW, Nembutal sodium solution, Abbott Laboratories, North Chicago, IL, USA) in 0.2 ml of saline and placed in supine position on the operative field. A 0.7∼1.0-cm incision in an area of the mouse back was made for implanting the mouse port. A subcutaneous pocket was made by insertion of hemostats (micro-mosquito hemostat, Fine Science Tools Inc., Foster City, CA, USA), and then the port was implanted into the pocket. Another skin incision (0.5 cm) was made in the neck area to expose a jugular vein, and a canal was created under the skin between two incisions using a straight forceps. The catheter connected to the port was held and dragged through the canal and was introduced into the jugular vein. The vein was isolated and secured with two 7-0 sutures. After surgery, each mouse was injected intramuscularly with 2 mg/kg BW of buprenorphine hydrochloride (Buprenex, Hospira Inc., Lake Forest, IL, USA) in 0.2 ml of saline for analgesia. The cage containing the mice was then placed on a SoftHeat Heating Pad (Kaz Inc., Southborough, MA, USA) for about 1 h during recovery from surgery. The port was rinsed with heparin (8.6 U/ml) in 0.1 ml of PBS (pH 7.4) every 24 h to prevent blood coagulation inside of the catheter. The lumen of the port had 0.05-ml dead volume, so the heparin solution was mixed with gadoteridol (0.0267 mmol/ml) to fill the dead volume with gadoteridol and avoid dilution.

Appendix B: Tumor Tissue Staining

Each tumor was sliced into two pieces and then immersed into 10% neutral-buffered formalin overnight at room temperature. Tissue sections of 5-µm thickness were cut on an Accu-Cut SRM microtome (Sakura, Tokyo, Japan). Sections of paraffin-embedded tissue were mounted on Bond-Rite slides from Richard-Allan Scientific (Kalamazoo, MI, USA) and heated at 60°C for 2 h. Paraffin was removed from the sections by three changes of xylene and rehydrated through graded alcohols from absolute to 70% for 5 min each.

Antigen retrieval was performed with high-temperature treatment with 0.5 M Tris buffer at pH 10. H₂O₂ avidin and biotin solutions and 3% goat serum were used to quench peroxidases, block endogenous biotin, and block nonspecific binding. Rabbit polyclonal antibody to CD31 (Abcam Inc., Cambridge, MA, USA) was diluted 1:200 and applied to the tissue at room temperature for 1 h. The secondary antibody was goat antirabbit (Jackson Immuno Research, West Grove, PA, USA) and the label was avidin–HRP (Signet Pathology Systems, Dedham, MA, USA). After the DAB chromagen (BioGenex, San Ramon, CA, USA) was applied, the tissues were counterstained with hematoxylin and the coverslips mounted with Permount.

The TUNEL assay was performed with a Chemicon International ApopTag Peroxidase In Situ Detection kit (Temecula, CA, USA). The slides were rehydrated as above and pretreated for 1 min in 10 mM glycine at pH 3 with fast cooling after heating in a pressure cooker. The slides were rinsed for a minimum of 2 h with deionized water after quenching with H₂O₂. The chromagen used was 3-3′diaminobenzidine according to the manufacturer’s protocol (BioGenex, San Ramon, CA, USA). After 7 min, the slides were rinsed with water and lightly counterstained with Mayer’s hematoxylin. The sections were dehydrated through graded alcohols, 70% to absolute ethanol, followed by three xylene rinses for 5 min each. The coverslips were mounted with Permount.