Determine transcriptomic changes in TGF-β–activated CAFs following Ce6@D4-EV–mediated photodynamic therapy (RNA-seq)

Solid tumors, including pancreatic ductal adenocarcinoma (PDAC), drive the activation of resident quiescent fibroblasts into cancer-associated fibroblasts (CAFs), fostering the development of dense desmoplastic stroma. This pathological remodeling restricts drug delivery and immune infiltration, exacerbating tumor progression. To disrupt this self-reinforcing cycle, we engineered a multifunctional therapeutic platform based on Midkine (MDK)-targeting nanobody (Nb)-functionalized extracellular vesicles (EVs) for precision photoimmunotherapy (PIT). The system leverages the overexpression of MDK in the tumor microenvironment (TME) to achieve selective targeting. In subcutaneous and orthotopic PDAC models, near-infrared (NIR) imaging confirmed the high tumor-targeting specificity of MDK-D4-EVs. Upon loading the clinically approved second-generation photosensitizer chlorine e6 (Ce6), Ce6@D4-EV mediated targeted photodynamic therapy (PDT) triggered immunogenic cell death (ICD), promoting dsDNA release from dying cancer cells and remodeling the immunosuppressive TME via cGAS-STING pathway activation in tumor-associated macrophages (TAMs). Concurrently, Ce6@D4-EV reprogrammed CAFs to suppress extracellular matrix (ECM) deposition, enhancing tumor vascular perfusion, alleviating hypoxia, and improving therapeutic agent penetration and immune cell infiltration. This dual modulation significantly augmented the efficacy of combined therapies, including immune checkpoint inhibitors (anti-PD-1 and anti-CD47 blockades), adoptive T cell therapy, and gemcitabine (Gem) chemotherapy, leading to marked survival benefits in preclinical models. Collectively, this engineered EV-based PIT platform offers a potent strategy to overcome stromal barriers in desmoplastic tumors and establishes a foundation for clinical translation of photoimmunotherapy strategies.