Characterization and functional analysis of a slow cycling stem cell-like subpopulation in pancreas adenocarcinoma

Jennifer L. Dembinski 0 Stefan Krauss 0 0 J. L. Dembinski (&) S. Krauss Section for Cellular and Genetic Therapy, Institute of Microbiology, Cancer Stem Cell Innovation Center (CAST), Rikshospitalet , Forskiningsparken, Gaustadalleen 21, 0349 Oslo, Norway Evidence suggests that multiple tumors, including pancreatic adenocarcinoma, display heterogeneity in parameters that are critical for tumor formation, progression and metastasis. Understanding heterogeneity in solid tumors is increasingly providing a plethora of new diagnostic and therapeutic approaches. In this study, a particular focus was put on identifying a subpopulation of stem cell-like, slow cycling tumor cells in a pancreas adenocarcinoma cell lines. Using a label retention technique a subpopulation of slow cycling cells (DiI?/SCC) was identified and further evaluated in the BxPC-3 and Panc03.27 cell lines. These slowly cycling cells managed to retain the lipophilic labeling dye DiI, while the bulk of the cells ([94%) did not. The DiI?/SCC population, showed only a partial overlap with the CSC markers CD24?/CD44?, CD133? and ALDH but they survived chemotherapeutic treatment, and were able to recreate the initial heterogeneous tumor cell population. DiI?/SCCs exhibited an increased invasive potential as compared with their non-label retaining, faster cycling cells (DiI-/FCC). They also had increased tumorigenic potential and morphological changes resembling cells that have undergone an epithelial to mesenchymal transition (EMT). Analysis of DiI?/SCC cells by real time PCR revealed a selective upregulation of tell tale components of the Hedgehog/TGFb pathways, as well as a down-regulation of EGFR, combined with a shift in crucial components implied in EMT. The presented findings offer an expanded mechanistic understanding that associates tumor initiating potential with cycling speed and EMT in pancreatic cancer cell lines. - Pancreatic cancer is known for its extensive local tumor invasion and early systemic dissemination. The molecular basis for these characteristics is not yet fully understood. Therefore, despite advances in surgical and medical therapy, little effect has been made on the mortality rate of this disease [1]. Most attempts to better understand the characteristics of pancreatic adenocarcinoma have focused on studying gene and protein expression profiles of total samples of pancreatic adenocarcinoma, and have not taken into account the heterogeneity of cells within a particular tumor. However, based on the concept of cancer stem cells (CSC), claims are made that the ability of a tumor to grow and propagate may depend on small subsets of cells, as only a minority of cancer cells showed extensive proliferation when examined for their proliferative potential in various in vitro or in vivo assays [24]. In particular cells that are either positive for the surface antigens CD24 and CD44, CD133 or display ALDH activity have been reported to exhibit increased self renewal and tumor initiating potential, as well as to give rise to renewed heterogeneity in pancreas adenocarcinoma [3 7]. Cells with such properties are termed by various authors CSC or tumor initiating cells. Interestingly, the CD24?/CD44? cells in pancreas adenocarcinoma also display increased expression of components of the sonic hedgehog (Shh) stem cell signaling pathway [3, 7]. However, the role of the Hh signaling pathway and its mediator, the zinc finger transcription factor Gli1 [8], in etiology and progression of pancreas adenocarcinoma is not fully understood, although there is evidence to support a role of the Hh pathway in this tumor. The central importance of Gli1 mediated signaling was confirmed by a broad genetic analysis of pancreatic cancer that identified genetic alterations in 100% of samples, together with changes in KRAS, TGF, and Wnt/Notch. [9]. All 4 pathways are central in developmental and stem cell biology where they control cell cycle, developmental potential, cell adhesiveness and apoptosis [8, 10]. Although the involvement of these pathways in tumors is well established, less is known about their role and involvement in tumor heterogeneity or subpopulations. Analysis of the correlation of pathways to subpopulations will be of substantial significance. It is believed that subpopulations of cells within the tumor microenvironment can undergo changes and are the main contributor to metastatic disease. Interestingly, a recent study where a chemo-resistant population of pancreatic cancer cells was created, suggested that the resistant population of cells had undergone EMT changes and was now of a more motile invasive phenotype as compared to the parental line [11]. EMT, as controlled by developmental pathways, is a key player in cancer metastasis as it allows cells to migrate and invade surrounding issues and escape into the bloodstream, en route to establishing metastasis. Once these metastatic cells reach their destination, they can undergo reverse EMTmesenchymal epithelial transition (MET), to establish secondary tumors. Consequently, EMT stimuli can generate cells with properties of stem or progenitor cells. In a recent report, normal mammary epithelial cells were induced to acquire the CD44high/CD24low breast CSC phenotype after exposure to TGFb [12]. This implies that expression of TGFb either converts CD44low/CD24high cells to the CD44high/ CD24low CSC phenotype, thereby suggesting that the majority of cells have the possibility to switch to this CSC phenotype; or that the EMT stimuli inhibits the proliferation of the epithelial tumor cell population and induces the proliferation or stops the differentiation of the CSC. As a result, it has been suggested that CSCs may actually not be distinct entities, but rather tumor cells that transiently acquire stem cell-like properties as a consequence of EMT [13]. Such transient subpopulations of cancer cells with stem cell character may not only have an increased innate ability to resist chemo and radio therapies [1416], but may carry a specific profile of developmental signals, which could make them amenable to therapeutic approaches through specific pathway antagonists. In this report, a subpopulation of DiI?/SCC in the pancreatic adenocarcinoma cell lines BxPC-3 and Panc03.27 was identified, selected upon and sorted by cell cycle speed. The resulting population (DiI?/SCC) was overlapping, but not identical, with the previously reported pancreatic population of tumor initiating cells (CD133?, ALDH?, or CD24?/CD44?). DiI?/SCCs were morphologically distinct and showed a more malignant, invasive and neoplastic phenotype when compared to the fast cycling bulk of cells (DiI-/FCC). Quantitative RT-PCR profiling confirmed that DiI?/SCC showed alterations in key developmental and stem cell signaling pathways, and an expression profile consistent with EMT conversion. Materials and methods Cells and culture conditions BxPC-3 and Panc03.27 pancreatic adenocarcinoma cel (...truncated)