EMT and Dissemination Precede Pancreatic Tumor Formation

1. Department of Biochemistry and Molecular Biology Reporter: Ma Ting (马婷) EMT and Dissemination Precede Pancreatic Tumor Formation DOI10.1016/j.cell.2011.11.025

2. Metastasis: the final step in a progressive” Darwinian” sequence?

3. Two clinical observation: • The appearance of metastatic lesions years after resection of small tumors with no clinically evident metastases at diagnosis • metastases of unknown primary, account for as many as 4–5% of all clinical metastases

4. Metastasis : an inherent feature of a tumor very early in its natural history? How to identify markers that distinguish cancer cells from normally residing cells in the bloodstream or at the sites of seeding? How to study EMT in vivo?

5. A lineage labeling system • Mouse models : PKCY mice Pdx1-Cre--Kras and P53 mutations--RosaYFP • Mouse models : IKCY mice Pdx1-Cre--Krasmutations--p16Ink4a/Arfdeletion--RosaYFP • Control mouse models: CY mice Pdx1-Cre; RosaYFP(CY) animals • Pdx1: only active in endoderm-derived pancreatic cells • P53、Kras: mutated with high frequency in human pancreatic cancers • Rosa YFP: track pancreatic epithelial cells during tumor progression, resulting in highly specific and efficient (>95%) labeling

6. Enhanced Detection of EMT using Epithelial Lineage Tracing Cells in the intermediate stage of EMT : Cells co-expressed an epithelial marker(E-cad) and a mesenchymalmarker (zeb1/Fsp1) -----10% O Cells completed an EMT : Cells co-expressed an epithelial marker(E-cad) and a mesenchymalmarker (zeb1/Fsp1)-----42% P、Q EMT was not detected in lineage-labeled CY control mice by either method L-N

7. EMT in Premalignant Lesions ”PanIN mice” :8- to 10-week-old PKCY ,only precancerous PanIN lesions and no histological evidence of carcinoma EMT was also prevalent in areas of acinar-to-ductalmetaplasia (ADM), particularly in ADMIs (acinar-to-ductalmetaplasia with Inflammation) EMT occurs in PanIN lesions and ADMIs prior to tumor formation.

8. Pancreatic Epithelial Cells Spread before Tumor Formation Individual YFP+ cells :“delamination” in PanIN 2 and 3 lesions. Most of these cells expressed zeb1 and had acquired a fibroblast-like morphology, making them indistinguishable from surrounding stromal cells by conventional histology Cells derived from the pancreatic epithelium are present in the circulation of mice with no evidence of carcinoma YFP+ circulating pancreatic cells (CPCs) were readily detected in the blood by flowcytometry in PDAC mice n E CPCs were also abundant in the bloodstream of PKCY PanINC-G carry the Gly→Asp mutation at codon 12 of the  KrascDNA

9. Pancreatic Epithelial Cells Spread before Tumor Formation • CPCs from PanIN mice might seed distant organs? 16/20 of PDAC mice have liver/lung metastasis 0 metastasis in PanIN mice but 4/11YFP+ cells seed in liver (Most were single cells lacated near blood vessels ,without zeb1 or E-cad) 0 YFP+ cells seed in liver in CY control mice

10. Characterization of CPCs The vast majority of CPCs do not survive passage through the pulmonary circulation There were no statistically significant differences in cell-surface phenotype between CPCs from the two groups  CPCs from PDAC and PanIN animals are phenotypically similar and that a large fraction maintains a mesenchymal phenotype in the circulation.

11. CPCs Exhibit Features of Cancer Stem Cells In human pancreatic tumors, cancer stem cells may be contained within a CD24+CD44+population of cells CPCs might exhibit features of CSCs

12. Cells that Have Undergone EMT Have Tumor-Initiating Properties

13. Inflammation Promotes EMT, Invasiveness, and Dissemination

14. Highlights of this research • Invading cells exhibit EMT in an autochthonous model of pancreatic cancer • Mutant cells enter the circulation before cancer is found on histology • Circulating pancreatic cells (CPCs) express cancer stem cell-associated markers • Inflammation is necessary and sufficient for EMT, invasion, and dissemination