Marker-Free Lineage Tracing Reveals an Environment-Instructed Clonogenic Hierarchy in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies. Effective treatment options are currently lacking, and targeted agents have so far demonstrated limited efficacy. It has been speculated that a rare population of cancer stem cells (CSCs) drives growth, therapy resistance, and rapid metastatic progression in PDAC. These CSCs are characterized by expression of genes associated with immature cells, and demonstrate high clonogenicity in vitro and tumorigenic potential in vivo. However, the unique relevance of CSCs in established PDAC tissue has not been determined. Here, we used a marker-independent stochastic clonal labeling system, in combination with a quantitative model of tumor expansion, to uncover the growth dynamics of PDAC tissue. We found that in situ all PDAC cells display clonogenic potential, which is in stark contrast to a strict hierarchical CSC model. Furthermore, we uncover that the proximity to activated cancer-associated fibroblasts determines which tumor cells actively drive tumor expansion. This means that the microenvironment is a dominant actor in defining the clonogenic activity of PDAC cells. Indeed, manipulating the PDAC associated stroma by Hedgehog pathway inhibition, altered the mode of tumor growth. These results reveal how tumor-stroma crosstalk shapes the growth dynamics and clonal architecture of these malignancies, and how perturbance of the stroma impacts on important aspects of PDAC biology.