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

Abstract

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 up-regulation of tell tale components of the Hedgehog/TGFbeta 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.

Fig. 1

Cell labeling, preparation and sorting a Schematic illustration of labeling, expansion and sorting of cells. b FACS analysis of BxPC-3 and Panc03.27 cells after 6 and 4 weeks growth, with 3.2 and 2.8% positive cells (DiI+/SCC), respectively

Fig. 2

Cell growth after DiI labeling. a Image of DiI+/SCC and DiI−/FCC cells after 4 (Panc03.27) or 6 (BxPC-3) weeks growth; before and after (BxPC-3) sorting (plated for high resolution imagery and taken after 24 h). b BxPC-3 normal growth (top) or with the addition of 100 μg/ml 5-FU (treated with 5-FU for 3 days) (bottom) of cells followed through 36 days. c Panc03.27 normal growth (top) or with the addition of 100 μg/ml 5-FU (treated with 5-FU for 3 days) (bottom) of cells followed through 22 days. d Growth of plated DiI+/SCC after sorting (times represent growth duration after plating). Scale bars represent 100 μm

Fig. 3

Invasion and Colony Forming Assays a Soft agar assay for colony formation. Graph represents results after 14 (Panc03.27) or 21 (BxPC-3) days of culture with DiI+/SCC or DiI−/FCC sorted populations. An increase in colony formation was seen with the DiI+/SCC population. b Invasion assay run with freshly sorted DiI+/SCC vs. DiI−/FCC populations. Results show increased invasive potential of the DiI+/SCC population over the DiI−/FCC population after 20 and 44 hours

Fig. 4

Expression of CSC markers a FACS analysis of percent CD24⁺/CD44⁺ cells in the bulk verses DiI+/SCC populations. b FACS analysis of percent CD133⁺ cells in the bulk and DiI+/SCC populations. c FACS analysis of percent ALDH⁺ cells in the bulk and DiI+/SCC populations

Fig. 5

Gene Expression Analysis a Change in gene expression, obtained from RT-PCR analysis, in DiI+/SCC as compared to DiI−/FCC populations. b RT-PCR profiling displaying return of gene expression levels to that of the original population after 30 (Panc03.27) or 60 (BxPC-3) days of repopulating growth by DiI+/SCC