Imaging Breast Cancer Metastasis over Endothelium to Bone

Bone is one of the common metastasis sites in patients with breast cancer. Once breast cancer cells reach bone, the result is devastating due to the vicious feedback cycle between cancer cells and bone stromal cells. But, for the cancer cells to get to the bone they must pass through the endothelium, which is normally covered by the glycocalyx; a thin layer rich in carbohydrates that protects endothelium against foreign objects, such as cancer. However, how bone metastasis is effected by the integrity of the glycocalyx is not well understood. Here, we created an in vitro system to mimic the interplay among cancer, endothelium, and bone seen in vivo. We did that by creating an endothelium (HUVEC) monolayer on top of a transwell insert (cat3464, Corning) with MDA-231 (1833 subclone) breast cancer cells seeded over the endothelium and bone derived condition medium below the transwell. The system was customized to enable direct imaging of the migration process of breast cancer cells through the endothelium. We also tested methods to impair the endothelial glycocalyx using heparinase III (Hep III) or IL-1beta. These impairments will be used to examine their effects on cancer migration in our future experiments. We concluded that (i) a tight monolayer can be achieved using the endothelial cells with a seeding density of 10,000 cell/mL and four days culture time; (ii) axial (xz) imaging of cancer-endothelium interactions is feasible using a regular inverted confocal; (iii) the optimal condition for glycocalyx impairments was .1U/mL for 2 hours using Hep III. We plan to use this in vitro system for live imaging breast cancer metastasis over endothelium to bone in both impaired and unimpaired glycocalyx systems.