Ultrasound-responsive oxygen-loaded microbubbles induce vascular normalization and immune modulation in pancreatic ductal adenocarcinoma.

Vascular normalization (VN) has been proposed as a strategy to restore tumor vascular function and promote antitumor immunity under anti-angiogenic conditions. However, conventional anti-angiogenic agents often cause excessive vessel pruning, which exacerbates subsequent tumor hypoxia and thereby limits the effectiveness of immunotherapy. This study evaluated whether oxygen-loaded microbubbles (OMBs) combined with focused ultrasound could induce tumor VN and activate antitumor immunity in a "cold" tumor model. Tumor perfusion and intratumoral oxygen tension were monitored following OMB treatment in a murine subcutaneous pancreatic ductal adenocarcinoma (Panc02) model. VN was assessed by vessel density, vessel maturity, and hypoxia-associated marker expression. Immune activation was evaluated by immunohistochemical detection of CD4⁺ and CD8⁺ T cells, intratumoral immune-related cytokines, and flow cytometric analysis of splenic T-cell populations. Primary and distant tumor growth were monitored to assess systemic antitumor effects. OMB treatment induced transient VN during days 2-4 after treatment without detectable changes in vessel density. This was associated with improved tumor perfusion and alleviation of hypoxia, facilitating increased intratumoral CD4⁺ and CD8⁺ T-cell infiltration. Systemic immune activation was evidenced by sustained expansion of splenic CD4⁺ and CD8⁺ T-cell populations through day 16. Distant tumor growth was significantly suppressed, consistent with VN-induced systemic antitumor immunity. These findings suggest that VN plays a critical role in enabling immune cell infiltration and activation in this "cold" tumor model. OMB treatment provides a means to transiently restore vascular function and modulate the tumor microenvironment, thereby promoting antitumor immunity.