315 Background: Notch is a family of cell surface receptors that has been demonstrated in several malignancies to regulate differentiation, proliferation, and metastasis. We have investigated the role of Notch signaling in bladder cancer and report here effects of modulating Notch2 in preclinical models of bladder cancer. Methods: Notch2 staining was examined in cystectomy patients by immunohistochemistry (IHC). The high NOTCH2 expressing UM-UC3 and UM-UC13 bladder cancer cells were stably transduced with either Notch2 specific or control shRNA. The low Notch2 expressing RT4V6 and RT112 cells were transduced with NOTCH2 intracellular domain or empty vector. A Notch2 inactivating antibody, NRR2Mab, was provided by Genentech, Inc. (San Francisco, CA). The effects of NOTCH2 overexpression and inhibition were assessed for changes in proliferation under both adherent (AH) and anchorage independent conditions (AI), and invasive activities in vitro, as well as for expression of markers of epithelial to mesenchymal transition (EMT) and stem cell markers. In vivo effects were evaluated in an orthotopic bladder cancer xenograft model with bioluminescence imaging. Results: NOTCH2 overexpression in IHC correlated with higher grade and stage. Both NOTCH2 overexpressing cells showed spindle shaped morphology and increased cell growth in AH and AI and invasion compared to mock cells. This was associated with cell cycle progression and increased expression of EMT and stem cell markers. Stable NOTCH2 knockdown cells and cells treated with NRR2Mab demonstrated decreased growth in AI and invasion, and this was associated with decreased expression of EMT and stem cell markers. In vivo, RT4V6 NOTCH2 overexpression increased xenograft tumor growth and NOTCH2 overexpression increased ki67 positive cells and promoted EMT in IHC. Stable Notch2 knockdown inhibited xenograft tumor growth in UM-UC3 and UM-UC13. NRR2Mab treatment also inhibited UM-UC13 xenograft tumor growth and lymph node metastasis. Conclusions: NOTCH2 plays a crucial role in growth, self-renewal and invasion in bladder cancer. These results provide pre-clinical proof of principle that Notch2 inhibition may be a rational treatment for bladder cancer.
Monitoring of Xenograft Tumor Growth and Response to Chemotherapy by Non-Invasive In Vivo Multispectral Fluorescence Imaging
