Formononetin from Jianpi Tongluo Formula Suppresses Colorectal Cancer Progression Via AXL/JAK2/STAT3 Axis-Mediated Inhibition of M2 Tumor-Associated Macrophage Polarization

Treatment of colorectal cancer (CRC) is limited by drug resistance and an immunosuppressive microenvironment. Tumor-associated macrophages (TAMs), especially M2-polarized TAMs, promote CRC progression. This study investigates the effects of formononetin (FN), a candidate bioactive constituent identified from Jianpi Tongluo Formula (JPTLF), on M2-TAM polarization and CRC progression. Multi-omics integration (network pharmacology, transcriptomics, weighted gene co-expression network analysis and single-cell sequencing) was used to identify key targets. Absorbed components were analyzed by ultra-performance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry. Molecular docking and dynamics simulations predicted compound-target interactions, and cellular thermal shift assays (CETSA) were performed to assess AXL thermal stability following FN treatment. In vitro assays, MC-38 xenograft models and patient-derived CRC organoids were employed for validation. AXL was identified as the sole overlapping target. FN, present in Astragalus membranaceus, Sophora flavescens and Glycyrrhiza uralensis, increased AXL thermal stability in CETSA, supporting FN–AXL target engagement. FN co-treatment with tumor-conditioned medium reduced M2 markers (CD206, arginase 1) and suppressed AXL, Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation. The AXL activator recombinant mouse Gas6 (rmGas6) partially reversed these effects, while the AXL inhibitor R428 mimicked FN’s action. In MC-38 xenografts, FN (100 mg/kg) inhibited tumor growth, reduced intratumoral M2-TAMs without observed body weight loss or histological injury in the major organs examined. Patient-derived CRC organoids were sensitive to FN. Formononetin suppresses CRC progression, with inhibition of M2-TAM polarization through AXL/JAK2/STAT3 signaling contributing to its antitumor activity. These findings provide preclinical support for further investigation of FN as a candidate compound for CRC treatment.