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

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.

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.

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).

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.

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 CD8⁺T cell functionality, modulating macrophage polarization, and reducing the abundance of malignant epithelial cells.

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.