Hedyotis diffusa Willd-Scutellaria barbata herbal pair suppresses triple-negative breast cancer metastasis by targeting the tumor-associated macrophage-driven OPN-CD44 signaling axis.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by a high propensity for metastatic relapse and a robust infiltration of tumor-associated macrophages (TAMs), which orchestrate a pro-metastatic microenvironment. Hedyotis diffusa Willd-Scutellaria barbata (HS), a classical herbal pair, exhibits potent antitumor activities; however, its specific role in modulating TAM-dependent metastasis remains unclear.

PURPOSE: This study investigated whether HS suppresses TNBC metastasis by modulating the functional state of TAMs, and sought to define the bioactive constituents and their molecular targets.

METHODS: The anti-metastatic activity of HS was assessed in a 4T1-luc orthotopic tumor model and a non-contact co-culture system. Macrophage depletion with clodronate liposomes was used to assess the contribution of macrophages to HS efficacy. Single-cell RNA sequencing analysis was performed to characterize macrophage-tumor cell communication. High-resolution LC-MS, molecular docking, surface plasmon resonance (SPR), and cellular thermal shift assays (CETSA) were integrated to identify HS derived constituents and validate their target engagement.

RESULTS: HS reduced orthotopic tumor growth and distant metastatic burden in vivo. HS also reduced F4/80 positive macrophage accumulation and CD206 positive TAM associated signals in tumor tissues, and suppressed IL-4/IL-13 induced macrophage polarization in vitro. In co-culture models, HS pretreated macrophages showed a reduced ability to promote TNBC cell migration, invasion, epithelial-mesenchymal transition, and clonogenic growth. Macrophage depletion attenuated the antitumor effects of HS and apigenin (API), supporting a macrophage-dependent mechanism. Single-cell analysis identified macrophage-associated OPN and its interaction with tumor-cell CD44 as a candidate paracrine signaling axis. HS reduced macrophage OPN expression and attenuated CD44-MEK/ERK signaling in TNBC cells. API was identified as an OPN-binding constituent of HS and disrupted macrophage-derived OPN-mediated CD44-MEK/ERK activation. In vivo, API suppressed orthotopic tumor growth and reduced lung metastatic lesions.

CONCLUSION: This study demonstrates that the HS herbal pair suppresses TNBC metastasis by remodeling the macrophage functional state and disrupting the signaling axis between OPN and CD44 driven by TAMs. The identification of API as an OPN binding constituent provides a mechanistic basis for further investigation of compounds derived from HS that target macrophage-tumor crosstalk in TNBC.