Abstract 2295: Succession of transiently active TIC clones drives long-term human pancreatic cancer progression .
Authors: Oppel Felix , Ball Claudia R. , Hoffmann Christopher M. , Dieter Sebastian M. , Dubash Taronish D. , Koch Moritz , Weitz Jürgen , Bergmann Frank , Schmidt Manfred , Abel Ulrich , von Kalle Christof , Glimm Hanno 
Published: August 2014

Abstract
Despite accumulating evidence indicating a pivotal role of tumor-initiating cells (TIC) in tumor progression and metastases formation of pancreatic cancer (PC), very little is known about the clonal TIC dynamics within PC tumors in vivo. It is unclear whether the high aggressiveness of PC is driven by a homogeneous population of self-renewing TIC or whether distinct classes or activation states of TIC drive long-term disease progression. To address this we tracked the clonal contribution of individual TIC to tumor formation in vivo.
Serum-free adherent cultures were established allowing enrichment of PC TIC without restriction to a certain phenotype. These cultures grow as 3-dimensional epithelial colonies with tight cell-to-cell contacts und reliably form subcutaneous tumors in NSG mice. To induce differentiation of TIC, FBS was added and FGF2, FGF10 and Nodal were withdrawn. Under these conditions, irregular shaped cells form a monolayer and down regulate markers previously described for TIC or normal pancreatic progenitors. Strikingly, despite profound changes in morphology and marker expression tumor initiation and self-renewal of TIC in serial transplantation were unchanged. Moreover, sorted CD133+ and CD133− cells equally efficient formed tumors containing similar proportions of CD133+ in vivo. Together these data indicate an unexpected phenotypic plasticity of TIC in PC.
To determine the clonal kinetics of individual self-renewing TIC in vivo, early passage serum-free cultures from 3 patients were transduced with a lentiviral vector (LV) and serially transplanted. LV stably integrate into the host cell genome resulting in TIC clone specific fusion sequences which were identified by highly sensitive LAM-PCR and high throughput sequencing. In primary mice, 0.003-0.113% of all transduced cells contributed actively to tumor formation. Unexpectedly, in subsequent generations tumor formation was mainly driven by distinct TIC clones not detectable in earlier but recruited to tumor formation in later generations. Moreover, individual primary xenografts generated pairs of secondary tumors with very little overlap between clonal compositions. Mathematical modeling indicates strong changes in proliferative activity of individual, otherwise homogenous TIC which predominantly produce non-tumorigenic progeny with very limited self-renewal.
These data indicate that long-term progression of PC is driven by a succession of transiently active TIC generating tumor cells in temporally restricted bursts. The recruitment of inactive TIC clones to tumor formation after serial transplantation points to a context-dependent regulation of TIC activity within the growing tumor. Further understanding the mechanisms regulating the balance between activated and resting states of TIC may allow developing specific treatment strategies targeting this most relevant cell population in PC.
Citation Format: Felix Oppel, Claudia R. Ball, Christopher M. Hoffmann, Sebastian M. Dieter, Taronish D. Dubash, Moritz Koch, Jürgen Weitz, Frank Bergmann, Manfred Schmidt, Ulrich Abel, Christof von Kalle, Hanno Glimm. Succession of transiently active TIC clones drives long-term human pancreatic cancer progression . [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 2295. doi:10.1158/1538-7445.AM2013-2295