Isolation of Breast cancer CTCs with multitargeted buoyant immunomicrobubbles

Circulating tumor cells (CTCs) are extremely rare cells found in blood of metastatic cancer patients. There is a need for inexpensive technologies for fast enrichment of CTCs from large blood volumes. Previous data showed that antibody-conjugated lipid shell immuno-microbubbles (MBs) bind and isolate cells from biological fluids by flotation. Here, blood-stable MBs targeted to several surface markers for isolation of breast tumor cells were developed. MBs coated with anti-human EpCAM antibodies showed efficient binding of EpCAM⁺ breast cancer cell lines SKBR-3, MCF-7, and MDA-MB-453, whereas anti-human EGFR MBs showed binding of EpCAM^LOW/NEGATIVE cell lines MDA-MB-231 and BT-549. Multitargeted anti-human EpCAM/EGFR MBs bound all cell lines with over 95% efficiency. Highly concentrated MB-bound tumor cells were collected in a microliter volume via an inverted vacuum-assisted harvesting setup. Using anti-EpCAM and/or anti-EpCAM/EGFR MBs, an efficient (70-90%) recovery and fast (30min) isolation of the above-mentioned cells and cell clusters was achieved from 7.5mL of spiked human blood. Using anti-EpCAM MBs and anti-EpCAM/EGFR MBs, cytokeratin-positive, CD45-negative CTCs were detected in 62.5% (10/16) of patients with metastatic breast cancer and CTC clusters were detected in 41.7% (5/12) of CTC-positive samples. Moreover, in some samples MBs isolated cytokeratin positive, CD45 negative tumor-derived microparticles. None of these structures were detected in blood from non-epithelial malignancies. The fast and inexpensive multitargeted platform for batch isolation of CTCs can promote research and clinical applications involving primary tumors and metastases.