Endothelial Notch1 Regulates High-dose Radiation Therapy (HDRT)-induced Endothelial-to-mesenchymal Transition (EbdMT)

Abstract

There is increasing evidence that high-dose radiation therapy (HDRT) induces endothelial-to-mesenchymal-transition (EdnMT) which promotes abnormal tumor vascularity and radioresistance. We have previously shown that HDRT induces Notch1 signaling in tumor endothelial cells. Here, we investigate if endothelial Notch1 regulates HDRT induced EdnMT. HUVEC monolayers, in vitro, were also treated with 0, 2, 8, and 12Gy and immunoblot was performed for Notch1, Jagged1, VE-Cadherin. HUVEC monolayers were also treated with a soluble Notch inhibitor, Notch1 decoy, to assess the effect of Notch inhibition on EdnMT. Human neuroblastoma cells NGP were engineered to express Fc(control) and Notch1 decoy. These cells were intrarenally injected to develop xenograft tumors. Tumors were treated with 0 Gy and 12 Gy single fraction doses of HDRT. Mice were sacked at 72 hr after HDRT. Tumor sections were subjected to double immunofluorescence staining for aSMA (mesenchymal marker) and endomucin (endothelial marker). We also assessed the effect of combining HDRT with Notch inhibition on macrophage infiltration into the tumor microenvironment by performing immunofluorescence staining for F480 (a marker for tumor associated macrophage), INOS (marker for M1 macrophage) and MRC1 (a marker for M2 macrophage). In HUVEC monolayers, HDRT doses of 8 Gy and 12 Gy profoundly increased Notch1 and Jagged1 protein levels after 72 hr. The increased level of Notch1 and Jagged1 was associated with the reduction of endothelial marker VE-Cadherin level. Notch1 decoy treatment inhibited reduction of VE-cadherin indicating Notch inhibition blocked EdnMT. In vivo in NGP xenograft mice model, increased colocalization of endomucin and αSMA was observed at 72 hr 12 Gy post-irradiation, when compared to nonirradiated tumors, indicating transition of ECs. Interestingly, Notch inhibition reduced colocalizing αSMA (+) endomucin (+) staining in Notch1decoy+12 Gy tumors. Quantification showed that while ∼4% and ∼3% of endomucin (+) vessels expressed αSMA, following Fc+0 Gy and Notch1decoy + 0 Gy treatments, respectively, ∼30% of the surviving vessels were αSMA (+) after Fc+12 Gy. Notch inhibition reduced αSMA staining in surviving vessels as ∼4% of the vessels were αSMA (+) in N11-24-decoy+12 Gy tumors. Increased F480 and MRC1 staining was observed in tumor treated with 12 Gy, compared to untreated tumors, while no change in INOS staining was noted. Notch1 decoy treatment significantly decreased F480 and MRC1 suggesting Notch blockade decreased HDRT induced M2 macrophage infiltration. Our data shows that Notch blockade inhibits HDRT-induced EdnMT. Our data further shows that HDRT increased tumor-promoting immune suppressive M2 macrophage infiltration. Combing HDRT with Notch blockade inhibits M2 macrophage infiltration into the tumor microenvironment and can be a potential therapeutic approach to minimize immunosuppressive immune response.