Abstract 4911: Aggressive variants isolated from human pancreatic cancer passaging in mice are enriched in stem cell markers and PEAK1 expression.
Authors: Metildi Cristina A. , Kaushal Sharmeela , Strnadel Jan , Wright Tracy , Kelber Jonathan A. , Klemke Richard L. , Hoffman Robert M. , Bouvet Michael 
Published: August 2014

Abstract
A more aggressive variants of the human pancreatic cancer cell line BxPC-3 were generated by serially passaging primary pancreatic tumors in nude mice. Changes in tumorigenicity, proliferation, stem cell markers, PEAK1 expression and survival were determined. Orthotopic mouse models of human pancreatic cancer were established by injecting 1x106 BxPC-3-RFP pancreatic cancer cells into the pancreas of nude mice. Primary pancreatic tumors were harvested from premorbid mice to establish cell lines. The established cell line (106) was orthotopically injected into another set of mice. This cycle was repeated five times. Cells (106) of the sixth serially passaged cell line (P6) were orthotopically implanted into mice and monitored with weekly whole body fluorescence imaging. FACS and qPCR analysis were performed on primary and metastatic lesions for stem cell markers and PEAK1 expression. In a second group of mice, the parental (P0) and P6 cell lines were injected subcutaneously at different cell numbers (102, 5 x 102, 103, 104, 105 cells) to determine the minimum cell number that would produce a tumor. At 6 weeks, the tumors were harvested and weighed. The BxPC-3-RFP P6 variant had more rapid primary tumor growth and shortened overall survival in mice compared to the parental cell line (52.2±SE3.1 days vs 115.4±SE10.2 days, respectively, p&lt;0.001). Median time to metastasis was shortened from 11 weeks to 3 weeks in the parental and P6 cell lines, respectively (p=0.002). Tumor take was more readily established in the P6 cell lines in which 5/5 mice subcutaneously injected with 102 cells had tumor growth at 6 weeks compared to 0/5 mice injected with 102 cells of the P0 cell line (p=0.025). The P6 cell line also resulted in larger tumors at 6 weeks compared to the P0 cell line (102.2±28.5 mg vs 11.0±4.1 mg, respectively, p=0.002). qPCR evaluation demonstrated enrichment of stem cell markers (CD24, CD44, EpCAM) and PEAK1 expression in the P6 cell line compared to P0 cell line, by 4.6-, 15-, 4.2- and 16.3-fold, respectively. FACS demonstrated an increase in CD24+/CD44+/EpCAM+ cells from 69% in the parental cell line to 97.1% in the P6 cell line. In conclusion, serial passaging of tumor allows for in vivo selection of a more aggressive variant of the human pancreatic cancer cell line BxPC-3 associated with an enrichment in stem cell markers and an increase in PEAK1 expression.
Citation Format: Cristina A. Metildi, Sharmeela Kaushal, Jan Strnadel, Tracy Wright, Jonathan A. Kelber, Richard L. Klemke, Robert M. Hoffman, Michael Bouvet. Aggressive variants isolated from human pancreatic cancer passaging in mice are enriched in stem cell markers and PEAK1 expression. [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 4911. doi:10.1158/1538-7445.AM2013-4911