Surface Marker Identification to Capture Live Circulating Tumor Cells in Metastatic Triple-Negative Breast Cancer.

Metastatic triple-negative breast cancer (TNBC) is highly aggressive and lacks targeted therapies. Circulating tumor cells (CTC) are invaluable for monitoring metastatic tumor progression and treatment response but are difficult to capture because of their rarity and heterogeneity. Surface-based staining for live CTCs is essential to preserve RNA quality in single cells, but current markers tend to perform poorly on more mesenchymal tumor cells such as TNBCs. To enhance live TNBC CTC detection, we developed a workflow for live CTC capture and single-cell RNA sequencing (scRNA-seq). Using a mouse model of metastatic TNBC, we identified four new CTC surface markers, AHNAK2, CAVIN1, ODR4, and TRIML2, which specifically stain tumor cells. Combining antibodies against these markers improved CTC detection rates in multiple TNBC mouse models and patient samples. Also, combining these new markers with traditional CTC surface markers enhanced detection sensitivity, achieving the highest CTC coverage. This approach identifies diverse CTC populations, while preserving RNA quality for scRNA-seq, which is essential for understanding and therapeutically targeting metastatic breast cancer. The use of these newly identified CTC markers significantly enhances both detection and live capture of CTCs, paving the way for more effective use of liquid biopsy to monitor patient prognosis and treatment response in clinical settings. SIGNIFICANCE: CTCs are a powerful indicator of cancer metastasis; however, their scarcity makes them difficult to isolate. Current markers favor epithelial CTCs over mesenchymal populations. Our workflow for live CTC capture and sequencing enables discovery of new markers for both epithelial and mesenchymal CTCs. When combined with existing markers, we improve live CTC capture for more holistic studies of the metastatic process and offer a scalable method for discovering CTC markers.