Oncolytic virus encoding decorin and the CD40 ligand boosts T-cell response and achieves a durable antitumor response...

Triple-negative breast cancer (TNBC) remains an aggressive disease with limited treatment options. Oncolytic adenoviruses represent a promising platform, but their efficacy as single agents is often insufficient. This study examined whether an oncolytic adenovirus co-delivering decorin (DCN) and CD40 ligand (CD40L), designated rAd.DCN.CD40L, could enhance antitumor activity and immune activation against TNBC. Recombinant adenoviruses expressing DCN (rAd.DCN), CD40L (rAd.CD40L), or both proteins were constructed and evaluated in breast cancer cells using cell viability, apoptosis, migration, invasion, and damage-associated molecular pattern (DAMP) release assays. Compared with rAd.DCN or rAd.CD40L alone, rAd.DCN.CD40L triggered greater apoptosis and tumor cell death and elicited more pronounced DAMP release. Co-culture experiments also revealed enhanced dendritic cell function following rAd.DCN.CD40L treatment. For in vivo evaluation, syngeneic 4T1 and EMT-6 subcutaneous tumor models and a 4T1 lung metastasis model were employed. rAd.DCN.CD40L substantially delayed tumor growth and improved survival in tumor-bearing mice. Flow cytometric and histological analyses further confirmed enhanced local and systemic T-cell responses, elevated CD8⁺ T-cell activity, and modulated inflammatory macrophage phenotypes. Mechanistically, western blot analysis showed that rAd.DCN.CD40L may contribute to extracellular matrix remodeling and the down-regulation of the expression of epithelial-mesenchymal transition (EMT)-associated genes. Notably, CD8⁺ T-cell depletion markedly attenuated the therapeutic efficacy of rAd.DCN.CD40L, demonstrating that CD8⁺ T cells are critical functional effectors of tumor control. Collectively, these findings indicate that a DCN- and CD40L-armed oncolytic adenovirus represents a promising combinatorial strategy for TNBC immunotherapy, and warrants further preclinical and clinical investigation.