Rapamycin-Induced Endothelial Cell Death and Tumor Vessel Thrombosis Potentiate Cytotoxic Therapy against Pancreatic Cancer

Abstract

Purpose: Despite current chemotherapies, pancreatic cancer remains an uncontrollable, rapidly progressive disease. Here, we tested an approach combining a recently described antiangiogenic drug, rapamycin, with standard gemcitabine cytotoxic therapy on human pancreatic tumor growth.Experimental Design: Tumor growth was assessed in rapamycin and gemcitabine-treated nude mice orthotopically injected with metastatic L3.6pl human pancreatic cancer cells. H&E staining was performed on tumors, along with Ki67 staining for cell proliferation and immunohistochemical terminal deoxynucleotidyl transferase-mediated nick end labeling and CD31 analysis. Rapamycin-treated tumor vessels were also directly examined in dorsal skin-fold chambers for blood flow after thrombosis induction. Cell death in human umbilical vein endothelial cells was assessed by flow cytometry after annexin-V staining.Results: Rapamycin therapy alone inhibited tumor growth and metastasis more than gemcitabine, with remarkable long-term tumor control when the drugs were combined. Mechanistically, H&E analysis revealed tumor vessel endothelium damage and thrombosis with rapamycin treatment. Indeed, dorsal skin-fold chamber analysis of rapamycin-treated tumors showed an increased susceptibility of tumor-specific vessels to thrombosis. Furthermore, terminal deoxynucleotidyl transferase-mediated nick end labeling/ CD31 double staining of orthotopic tumors demonstrated apoptotic endothelial cells with rapamycin treatment, which also occurred with human umbilical vein endothelial cells in vitro. In contrast, gemcitabine was not antiangiogenic and, despite its known cytotoxicity, did not reduce proliferation in orthotopic tumors; nevertheless, rapamycin did reduce tumor proliferation.Conclusions: Our data suggest a novel mechanism whereby rapamycin targets pancreatic tumor endothelium for destruction and thrombosis. We propose that rapamycin-based vascular targeting not only reduces tumor vascularization, it decreases the number of proliferating tumor cells to be destroyed by gemcitabine, thus introducing a new, clinically feasible strategy against pancreatic cancer.

Rapamycin inhibits multiple stages of c-Neu/ErbB2–induced tumor progression in a transgenic mouse model of HER2-positive breast cancer

“…The rapid and focal nature of tumor cell death may reflect acute changes in established blood flow rather than attenuation of vessel proliferation. Rapamycin can promote thrombotic occlusion of tumor vessels (46,47). We observed histologic evidence of vessel occlusion, including clogged and dilated vessels and dying cells with features of ischemic necrosis, in tumors and peritumoral regions that were similar to those described for other types of tumors treated with rapamycin (data not shown).…”

Dual EGFR and mTOR targeting in squamous cell carcinoma models, and development of early markers of efficacy

“…A recent study in SCC cell lines demonstrates that inhibition of mTOR induces activation of Akt and results in antitumor effects in this tumour type (Amornphimoltham et al, 2005). In this model, as well as in other disease types such as pancreas cancer (Bruns et al, 2004), the antitumor effects of mTOR inhibitors appear to be in part related to their antiangiogenic effects. In our previous work, we have determined the susceptibility of a set of SCC cell lines both in vitro and in vivo to EGFR inhibitors (Amador et al, 2004;Jimeno et al, 2006).…”

“…It is known that mTOR inhibitors work, at least in part, by decreasing angiogenesis (Bruns et al, 2004;Stephan et al, 2004). Western blots.…”

In vivo antitumor effect of the mTOR inhibitor CCI‐779 and gemcitabine in xenograft models of human pancreatic cancer

“…Moreover, other investigators have demonstrated synergistic growth-inhibitory effects by combined treatment of gemcitabine and rapamycin in another orthotopic model of L3.6pl pancreatic cancer cells. 43 They have revealed that rapamycin induced apoptosis of endothelial cells and tumor vessel thrombosis in this model and suggested that combining rapamycin with other cytotoxic drugs could be a feasible therapy of pancreatic cancer. From these data, we speculate that an antiangiogenic effect of CCI-779 might be involved in the mechanism of its synergistic antitumor activity in this study.…”