Abstract

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.