To explore the effects and mechanisms of Arenobufagin on colorectal cancer using single-cell transcriptomics and organoid models.

Single-cell sequencing on colorectal cancer xenografts treated with Arenobufagin and phosphate buffered saline（PBS）was performed. The study analyzed the reduction in specific tumor cell subpopulations and their biological functions. EDU experiments assessed the impact of Arenobufagin on HCT 116 cell proliferation，and Western Blotting verified changes in protein expressions related to the Notch pathway. Organoid models were evaluated using Hematoxylin-Eosin and Immunohistochemistry staining.

The single-cell RNA sequencing results revealed that the proportion of the second tumor cell subpopulation significantly decreased following intervention. The subpopulation accounted for 21.57% in the PBS group and 14.16% in the Are group. Gene enrichment analysis indicated that these tumor cells were associated with biological processes such as protein transport，transcription regulated by RNA polymerase II，vesicle-mediated transport，negative regulation of transcription from DNA templates，vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus，apoptosis，tumor necrosis factor（TNF）signaling pathway，proteoglycans in cancer，ubiquitin-mediated protein degradation，cancer pathways，Notch signaling pathway，and Wnt signaling pathway. The EDU assay demonstrated that arenobufagin（Are）dose-dependently inhibited the proliferation of HCT116 cells within the concentration range of 100 nm to 300 nm compared to the PBSgroup. Western blot analysis showed that，compared to the PBSgroup，arenobufagin significantly increased the expression of BAX while downregulating the expression of BCL-2. Additionally，proteins associated with the Notch pathway，including Notch1，Jagged1，HES1，Cyclin D1，and DLL4，exhibited varying degrees of expression inhibition.

Arenobufagin effectively inhibits the proliferation and migration of colorectal cancer cells，likely through the suppression of the Notch signaling pathway.