1.06 Stromal activation mediates metastatic outgrowth of pancreatic cancer cells in the liver

W. Lo1, E. Van Beek1, S. Sinha1, A. Ranjan1, G. Chakraborty2, J. Davis1, R. T. Ripley1, J. Hernandez1  1National Cancer Institute,Thoracic And GI Oncology Branch,Bethesda, MD, USA 2Memorial Sloan-Kettering Cancer Center,Department Of Medicine,New York, NY, USA

Introduction:  Recent studies have suggested that metastasis is initiated by a subpopulation of cancer stem-like cells or by cancer cells that revert to the stem-like fate upon invasion into the stroma of the target organ. Like normal adult stem cells, the metastasis-initiating cells appear to enter quiescence and undergo reactivation in response to niche signals that are part of the extracellular matrix, embedded in it, or exposed on the surface of adjacent stromal or epithelial cells. The signaling cascades that mediate metastatic reactivation from quiescence remain poorly defined.    

Methods:  In order to evaluate the signaling mechanisms that differentiate those cells capable of metastatic outgrowth, we designed and implemented an innovative negative selection method. Specifically, we isolated dormant variants of the KPC tumor cells (from Pdx-1-Cre, KrasG12DLSL, Trp53R172HLSL mice) that lack the ability to form liver metastases following splenic injection. We then compared the transcriptomes, secretomes, and exosomes of dormant and fully metastatic cells. Next, we serially examined mouse livers at defined time-points after splenic injection of cells using immunohistochemical and immunofluorescence techniques. Finally, sphere-forming assays were undertaken with and without the addition of extra-cellular matrix proteins. Statistical analysis was undertaken using GraphPad Prism where appropriate. 

Results: Comparison of the transcriptomes, secretomes, and exosomes of dormant and fully metastatic cells indicated that the metastatic cells produce and package into exosomes cytokines and matrix proteins able to activate TGF-β signaling. In addition, we have observed that nascent micrometastases coopt the periportal cells and undergo expansion in close apposition with the abluminal surface of blood vessel, which results in vascular occlusion reminiscent of external compression. Intriguingly, the nascent tumors activate local collagen-producing cells before the growing tumor mass egresses and invades/replaces normal hepatic parenchyma. Analysis of an isolated “escape” dormant clone (revertant phenotype), which acquired the capability of forming liver metastasis upon splenic injection, revealed secretion of collagen cross-linking enzymes Loxl-2 and Loxl-3 similar to fully metastatic cells. Accordingly, metastatic cells, but not the dormant cells, were able to augment sphere-forming capacity in the presence of collagen, but no other ECM proteins. 

Conclusion: Stromal activation appears to be one of the earliest steps in liver colonization for metastatic pancreatic cancer. Interrupting TGF-β signaling and/or matrix stiffening through collagen cross-linking may be a reasonable strategy for adjuvant therapy after resection of pancreatic cancer.