Serial single-cell RNA sequencing unveils drug resistance and metastatic traits in stage IV breast cancer

Background: Drug resistance and metastasis are complex processes that remain poorly understood on the molecular level with single-cell resolution. We profiled the transcriptomic changes at the single-cell level associated with cancer cell progression, chemotherapy resistance, and metastasis in samples from a single de novo Stage IV breast cancer patient. Method: Pretreatment biopsy samples and posttreatment surgical specimens from the primary tumor and distant metastases were collected for single-cell RNA sequencing and subsequent cell clustering, copy number variation (CNV) estimation, transcription factor activity estimation, and pseudotime analyses. Results: CNV estimation analysis identified a small pretreatment cancer cell population that resisted chemotherapy and subsequently expanded. In the posttreatment primary tumors, new clones emerged, which we refer to as potential Metastatic Precursor Cells (MPCs), exhibiting the CNV profiles similar to metastatic cells. MPCs appeared to originate from hypoxic regions in the necrotic core of the tumor and exhibited expression profiles indicative of epithelial–mesenchymal transition. Comparison of MPCs to metastatic cancer cells also revealed dynamic changes in transcription factor activities and calcitonin pathway gene expressions. Conclusion: These findings demonstrate the utility of single-patient clinical sample analysis for clonal adaptations underlying tumor drug resistance, regrowth, and metastasis.