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 (CTCs) are invaluable for monitoring metastatic tumor progression and treatment response but are difficult to capture due to 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 (scRNAseq). Using a mouse model of metastatic TNBC, we identified four new CTC surface markers, AHNAK2, CAVIN1, ODR4, and TRIML2, that 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 scRNAseq, which is essential for understanding and therapeutically targeting metastatic breast cancer. Overall design: Single cell sequencing via SMARTseq2 of MDA-MB-231-LM2 cell culture cells and circulating tumor cells (CTCs) isolated from mice implanted with MDA-MB-231-LM2 cells in the mammary fat pad and allowed to spontaneously metastasize.