4.6. Circulating Tumor Cells (CTCs) Isolation and Culture

Blood samples were collected from advanced breast cancer patients according to the IRB protocol approved by the University of Texas Health Science Center at San Antonio (CTRC# 07-32). About 15 mL of Ficoll (GE Healthcare Life Sciences) was placed in a 50 mL conical tube, and 8 mL of the blood sample was carefully added on the top of the Ficoll. CTCs remained in the interphase peripheral blood mononuclear cells (PBMCs) layer after centrifugation at 400× g at RT for 30 min. The PBMC layer was carefully moved into a 15 mL conical tube and mixed with 10 mL of the MojoSort buffer (Biolegend, San Diego, CA, USA). After centrifugation again at 200× g for 5 min at 4 °C, CTCs were cleaned by the MojoSort buffer. Then, CTCs were cultured and expanded in the PRIME-XV tumorsphere medium (Fujifilm Irvine Scientific, Santa Clara, CA, USA) for 1 week for enrichment. For collection, cells were dispersed and washed by the MojoSort buffer and contaminated blood cell depletion was carried out using 10 μL of Human CD45-nanobeads (Biolegend, San Diego, CA, USA). CD45⁻ CTCs were harvested in the supernatant by a magnetic stand after a few washes and resuspensions with the MojoSort buffer.