To better understand the mechanism responsible for enhanced drug distribution by L-PDT, we assessed the real time changes of tumor/lung interstitial fluid pressure (IFP) and tumor blood flow (TBF) in mouse models of malignant pleural mesothelioma (MPM), lung adenocarcinoma, and pleural sarcomas in rodents before and during L-PDT. We found that photo-induction caused a significant decrease in IFP in all tumor models compared to controls while keeping TBF stable. These observations suggest photo-induction related tumor vasculature alterations with enhancement in convection between the intra- and extravascular spaces which improves drug distribution. The changes in IFP and TBF were similar to those observed with low-dose anti-angiogenic therapy (vascular normalization). Interestingly, no changes in IFP, TBF or drug distribution was observed in adjacent normal lung tissue equally submitted to photo-induction.

To further understand how L-PDT was stabilizing tumor vessels, we performed in vitro endothelial/pericyte co-culture assays where cells were exposed to L-PDT. We found that L-PDT caused a specific cytoskeletal remodeling in pericytes through RhoA, MLC and FAK. This was confirmed in MPM tumors in mice, where the same pathways, as well as enhanced pericyte coverage of endothelial cells and vascular stabilization after L-PDT, were detected.

Given the favorable impact of L-PDT on drug distribution but also its impact on the immune response, we tested whether L-PDT could enhance the immune response against MPM in a murine immunocompetent model and assessed how L-PDT affected subsequent immune checkpoint inhibition therapy. FACS analysis of L-PDT treated MPM (AE17 tumors grown in C57B6 mice) showed that a single PDT treatment enhanced the ratio of CD8+ cells over CD4 helper cells and increased the activity of antigen presenting cells (macrophages and dendritic cells) at day 7. While this had no impact on tumor growth, the combination of L-PDT with anti-CTLA-4 immune checkpoint therapy controlled MPM tumor growth significantly better than controls. Interestingly, the combination of L-PDT with anti-CTLA-4 caused a complete tumor regression in 30% of animals. Finally, enhanced E-Selectin expression, a molecule involved in leucocyte diapedesis, was observed at the endothelial level