Daisuke Shiokawa

The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain–containing ion transport regulator 3 (FXYD3), a component of the Na⁺/K⁺ pump. Accordingly, FXYD3⁺ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3⁺ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3⁺ CSCs were sensitive to senolytic Na⁺/K⁺ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3⁺ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na⁺/K⁺ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.

Intra-tumor heterogeneity (ITH) encompasses cellular differences in tumors and is related to clinical outcomes, such as drug resistance. However, little is known about the dynamics of ITH, owing to the lack of time-series analysis at the single-cell level. We performed single-cell exome and transcriptome sequencing of 200 cells and investigated how ITH is generated from one single cell in a mouse colorectal cancer model. The ITH of the transcriptome increased after transplantation from cultured organoids, while that of the exome decreased. Mutations generated in the culture did not greatly change at the transplantation at the bulk-cell level. The RNA ITH increase was due to the emergence of new transcriptional subpopulations. In contrast to the initial cells expressing mesenchymal-marker genes, new subpopulations repressed these genes at transplantation. Analyses of colorectal cancer data from The Cancer Genome Atlas revealed a high proportion of metastatic cases in human subjects with expression patterns similar to the new cell subpopulations in mouse. These results suggest that the birth of transcriptional subpopulations may be a key for adaptation to drastic micro-environmental changes when cancer cells have sufficient genetic alterations at later tumor stages. This study revealed an evolutionary dynamics of single-cell RNA and DNA changes in tumor progression, giving insights into the mesenchymal-epithelial transformation of tumor cells at metastasis in colorectal cancer.