Formononetin From Traditional Chinese Formula Shows Preclinical Activity Against Colorectal Cancer

Formononetin, a bioactive compound identified in the traditional Chinese medicine Jianpi Tongluo Formula, has shown the ability to suppress colorectal cancer progression in preclinical models by interfering with the polarization of tumor-associated macrophages, according to a study published in Biological Procedures Online on 14 October 2026.

Treatment of colorectal cancer remains limited by drug resistance and by the immunosuppressive microenvironment that tumors create around themselves. Tumor-associated macrophages, and especially their M2-polarized form, are known to promote colorectal cancer progression, making them an attractive target for therapeutic intervention.

To identify the compound and its molecular targets, the researchers integrated multiple analytical approaches: network pharmacology, transcriptomics, weighted gene co-expression network analysis and single-cell sequencing. Absorbed components of the formula were analyzed by ultra-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry.

Formononetin itself was found to be present in three herbs used in the formula: Astragalus membranaceus, Sophora flavescens and Glycyrrhiza uralensis.

Formononetin increased the thermal stability of AXL, supporting direct target engagement. Molecular docking and molecular dynamics simulations were used to predict the compound-target interaction before this experimental validation.

Functionally, when macrophages were co-treated with formononetin and tumor-conditioned medium, the compound reduced the expression of M2 markers, including CD206 and arginase 1, indicating a shift away from the immunosuppressive M2 phenotype. At the signaling level, formononetin suppressed the phosphorylation of AXL, Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), the axis through which AXL activity is transmitted into the cell.

The researchers used pharmacological tools to test causality in this pathway. Recombinant mouse Gas6, an AXL activator, partially reversed formononetin’s effects, while R428, an AXL inhibitor, mimicked the compound’s action. Together, these experiments support the conclusion that formononetin acts through inhibition of the AXL/JAK2/STAT3 axis to block M2 polarization of tumor-associated macrophages.

At a dose of 100 mg/kg, formononetin inhibited tumor growth and reduced the abundance of intratumoral M2 tumor-associated macrophages. No body weight loss or histological injury was observed in the major organs examined, an early indication of tolerability, although the authors note this applies only to the organs assessed.

The authors emphasize that these findings provide preclinical support for further investigation of formononetin as a candidate compound for colorectal cancer treatment. As the work combines cell culture, animal models and patient-derived organoids but has not yet been tested in clinical trials, its conclusions describe laboratory and preclinical activity rather than established therapeutic benefit in patients. The article is published open access, and this version is shared early as peer-reviewed, accepted research that remains subject to further editorial edits before the final Version of Record appears.

Subject of Research: Preclinical evaluation of formononetin from Jianpi Tongluo Formula as an inhibitor of M2 tumor-associated macrophage polarization in colorectal cancer

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

Article References: Yan, Y., Li, Y., Zhang, H., Zhao, Y., Liu, X., & Ren, J. (2026). Formononetin from Jianpi Tongluo Formula Suppresses Colorectal Cancer Progression Via AXL/JAK2/STAT3 Axis-Mediated Inhibition of M2 Tumor-Associated Macrophage Polarization. Biological Procedures Online. https://doi.org/10.1186/s12575-026-00364-y

Image Credits: AI Generated

DOI: 10.1186/s12575-026-00364-y

Keywords: formononetin, colorectal cancer, tumor-associated macrophages, M2 polarization, AXL, JAK2, STAT3, Jianpi Tongluo Formula, network pharmacology, organoids, xenograft model, preclinical research