Xian-Lian-Jie-Du Decoction suppresses colorectal cancer liver metastasis by inhibiting Plaur/Hbegf/EGFR-mediated actin cytoskeleton remodeling.

BACKGROUND: Colorectal cancer liver metastasis (CRLM) remains a major clinical challenge. Xian-Lian-Jie-Du Decoction (XLJDD) has shown anti-tumor activity against colorectal cancer, but its mechanism in CRLM remains unclear.

PURPOSE: To clarify the anti-metastatic mechanism of XLJDD and identify its Plaur-targeting active constituents in CRLM.

STUDY DESIGN: An integrated in vivo, in vitro, transcriptomic, proteomic, and single-cell mechanistic study.

METHODS: A CRLM mouse model was established by intrasplenic injection of CT26 cells. Transcriptomic and proteomic analyses were integrated to identify candidate targets. Single-cell RNA sequencing (scRNA-seq) was used to characterize tumor microenvironment changes. Wound-healing, Transwell, phalloidin staining, and an in vivo Plaur-overexpression rescue model were used for functional validation. XLJDD granules and XLJDD-derived compounds in biological samples were profiled by UPLC-MS/MS, and their binding to Plaur was examined by molecular docking, surface plasmon resonance (SPR), and microscale thermophoresis (MST). Hepatic exposure of cyclogalegenin was further quantified by targeted UHPLC-MRM-MS/MS.

RESULTS: XLJDD reduced hepatic metastatic burden in metastatic liver tissues. Integrated transcriptomic and proteomic analyses identified the Plaur/Hbegf axis as a candidate target of XLJDD. Mechanistically, Plaur promoted Mmp9-associated Hbegf release, whereas XLJDD suppressed EGFR/ERK signaling and reduced the expression of cytoskeleton-related effectors, including Fscn1 and Cdc42ep1. scRNA-seq revealed that XLJDD reduced a Plaur-high Car6+ malignant subpopulation, increased an Ifit1+ interferon-reactive state, and weakened Spp1-Cd44 signaling between malignant cells and Thbs1+ tumor-associated macrophages. In vivo, Plaur overexpression weakened the anti-metastatic effect of XLJDD. Cyclogalegenin was identified as a tissue-available constituent of XLJDD that directly binds Plaur and suppresses Plaur expression in CT26 cells. Targeted quantification confirmed the presence of cyclogalegenin in liver metastatic tissues after oral XLJDD administration.

CONCLUSION: XLJDD suppresses CRLM by targeting Plaur, restraining cytoskeleton remodeling, and remodeling the metastatic tumor microenvironment. Cyclogalegenin may contribute to this activity as a direct Plaur-binding constituent.