Notch3/4 Knockdown Inhibits Colon Adenocarcinoma Progression by Suppressing Tumor Cell Activity and Orchestrating VEGFA-Dependent Tumor Immune Microenvironment.

BACKGROUND: Colon adenocarcinoma (COAD) is a prevalent gastrointestinal malignancy characterized by dysregulation of multiple cellular signaling pathways, including the Notch pathway. However, the specific biological roles of Notch3 and Notch4 in COAD remain incompletely characterized.

METHODS: We aimed to investigate the effects of Notch3 and Notch4 downregulation on the tumor immune microenvironment and angiogenesis in COAD. Using a combination of in vitro and in vivo models, we evaluated the impacts of Notch3/4 interference on cell proliferation, migration, immune cell infiltration, and tumor progression.

RESULTS: Interfering with Notch3 and 4 significantly suppressed subcutaneous tumor progression. Notably, this inhibition was not only associated with reduced cell viability, but also with an altered immune environment characterized by enhanced macrophage M1 polarization and T lymphocyte activation, which may be linked to the c-Myc/VEGFA signaling axis but requires further mechanistic validation. Furthermore, the combination of regorafenib and Notch3/4 knockout (KO) exerted enhanced anti-tumor effects. More importantly, Mol_2, a candidate compound with predicted binding affinity for both Notch3 and Notch4, was screened out to significantly reduce the protein levels of Notch3 and Notch4 and tumor progression.

CONCLUSIONS: Our findings suggest that targeting Notch3 and Notch4 receptors may have therapeutic potential for modulating the tumor microenvironment and suppressing tumor progression in COAD.