Integrating CyTOF with scRNA-seq reveals that Cyclovirobuxine D inhibits HCC progression through hepatic immune microenvironment remodeling.

[BACKGROUND] Hepatocellular carcinoma (HCC) represents the predominant type of primary malignant liver cancer, characterized by high morbidity and mortality rates. The steroidal alkaloid cyclovirobuxine D (CVB-D), which is extracted from Buxus microphylla, has garnered increasing attention for its potential in anti-tumor research. Despite this, the immunoregulatory effects of CVB-D in HCC remain insufficiently explored.

[PURPOSE] This study investigated the role and underlying mechanisms of CVB-D in inhibiting HCC progression, aiming to identify potential therapeutic targets and provide new scientific evidence for HCC treatment.

[STUDY DESIGN] CVB-D was administrated to orthotopic HCC mouse models. The effects of CVB-D on the tumor immune microenvironment were systematically assessed via integrated cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq).

[METHODS] An orthotopic HCC model was generated in C57BL/6 mice via Hepa1-6 cell implantation, followed by intraperitoneal administration of different doses of CVB-D. Following a 14-day treatment period, fresh tumor tissues were collected for CyTOF and scRNA-seq analyses. Multiplexed immunofluorescence staining was performed on liver tissue sections to validate the in suit localization of key biomarkers. Serum cytokine levels were evaluated utilizing the Quantibody® Mouse Inflammation Array Kit.

[RESULTS] This research demonstrated that administration of CVB-D in orthotopic HCC mouse models notably inhibited tumor progression. Comprehensive analyses combining CyTOF and scRNA-seq demonstrated that CVB-D remodeled the hepatic immune microenvironment by enhancing CD8T cell functionality, modulating macrophage polarization, and reducing the abundance of malignant epithelial cells.

[CONCLUSION] These findings highlight the therapeutic promise of CVB-D as a novel candidate for HCC treatment, offering new insights into the development of innovative anti-cancer therapies.