Pancreatic cancer spheres are more than just aggregates of stem marker positive cells

Pancreatic cancer stem-like cells are described to be characterized by the membrane expression of CD24, CD44 and ESA, and their capacity to grow as spheres in serum-free medium containing well-defined growth factors. The capacity of a panel of 4 pancreatic cancer cell lines (PANC-1, CFPAC-1, PancTu-1, PSN-1) to form spheres was tested. All cell lines with the exception of PancTu-1 developed spheres. Phenotypically, the sphere-growing cells demonstrated an increased in vitro invasion capability. Both gene and protein expression of markers of metastases, (CXCR4, Osteopontin, CD44v6) and components of active Hedgehog pathway signaling were assessed. Spheres clearly demonstrated increased expression of the above mentioned markers when compared to their adherent counterpart. With the aim to identify a minimum set of markers able to separate cells that have the capacity to form spheres from those incapable, a principal component analysis (PCA) of the multidimensional dataset was performed. Although PCA of the 'accepted' stemness genes was unable to separate sphere-forming from sphere-incapable cell lines, the addition of the 'aggressiveness' marker CD44v6 allowed a clear differentiation. Moreover, inoculation of the spheres and the adherent cells in vivo confirmed the superior aggressiveness (proliferation, metastasis) of the spheres over the adherent cells. In conclusion, our study suggests that the sphere-growing cell population is not only composed of cells displaying classical stem membrane markers but needs CD44v6 positive cells to successfully form spheres. Our results also emphasize the potential therapeutic importance of pathways such as CXCR4 and Hedgehog for pancreatic cancer treatment.