Abstract
Abstract Purpose: The Notch signaling pathway has been extensively studied for its role in tumor angiogenesis but its function in tumor metastasis is largely elusive. In the present study we investigated the role of a notch receptor, Notch-1, in the regulation of neuroblastoma liver metastatic process. Methods: To investigate the function of Notch-1 in neuroblastoma liver metastasis we first generated immunodeficient (rag2gammaC deletion) Notch-1 heterozygous (+/-) mice. We crossed a Notch1 heterozygous (+/-) mouse with a rag2/gamma-c double knockout (-/-) mouse. The mouse with Notch1(+/-)rag2(+/-)gammaC(+/-) genotype was then backcrossed with a rag2gammaC double knockout (-/-) mouse to generate rag2(-/-)gammaC(-/-)Notch1(+/-) mouse or rag2(-/-)gammaC(-/-)Notch1(+/+)control mouse . These mice (n=5) were intracardically injected with 105 neuroblastoma NGP (MYCN amplified) cells expressing Firefly luciferase. The mice were subjected to IVIS- bioluminescence imaging, once in a week, to monitor the metastatic spread. All mice were sacrificed at week 8. At the time of sacrifice, mice were injected with Luciferin, sacrificed and liver was then dissected out, imaged and bioluminescence flux was measured (ex vivo imaging). The liver tissues were also sectioned (5um, paraffin) and H&E stained at multiple levels to further confirm the presence of metastases. Result: Intracardically injected NGP cells formed increased liver metastases in rag2(-/-)gammaC(-/-)Notch1(+/-) mice compared to littermate control rag2(-/-)gammaC(-/-)Notch1(+/+) mice. 4 out of 5 (80%) mice in Notch-het group and only 1 out of 5 (20%) mice in control group developed hepatic metastases. Notch-het livers showed multiple metastatic lesions with diameter ranged from 1-5 mm. Ex vivo bioluminescence imaging showed total flux (photon/sec) of Notch1 (+/-) livers were significantly higher (p<0.01) compared to Notch1 (+/+) control livers. Immunohistochemical staining against blood vessel markers such as Collagen IV, endomucin (endothelial cells) and aSMA (pericyte) showed extensive network of angiogenesis in Notch1 (+/-) liver metastases. Conclusion: Deficiency of Notch1 signaling in liver microenvironment presented an increase in metastases in Notch-het mice in NGP intracardiac injection metastatic model. Therefore our study indicates that Notch1 acts as tumor suppressor in neuroblastoma liver metastasis. Citation Format: Debarshi Banerjee, Alejandro Garcia, John Andrews, Emily Sbiroli, Jessica J. Kandel, Darrel J. Yamashiro. Deficiency of Notch-1 signaling in liver increases neuroblastoma hepatic metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2809. doi:10.1158/1538-7445.AM2013-2809
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