EMT and dissemination precede pancreatic tumor formation

Abstract

Metastasis is the leading cause of cancer-associated death but has been difficult to study because it involves a series of rare, stochastic events. To capture these events, we developed a sensitive method to tag and track pancreatic epithelial cells in a mouse model of pancreatic cancer. Tagged cells invaded and entered the bloodstream unexpectedly early, before frank malignancy could be detected by rigorous histologic analysis; this behavior was widely associated with epithelial-to-mesenchymal transition (EMT). Circulating pancreatic cells maintained a mesenchymal phenotype, exhibited stem cell properties, and seeded the liver. EMT and invasiveness were most abundant at inflammatory foci, and induction of pancreatitis increased the number of circulating pancreatic cells. Conversely, treatment with the immunosuppressive agent dexamethasone abolished dissemination. These results provide insight into the earliest events of cellular invasion in situ and suggest that inflammation enhances cancer progression in part by facilitating EMT and entry into the circulation.

Figure 1. Lineage labeled mouse models of pancreatic cancer and detection of EMT

(A) Schematic of the PKCY mouse model used in this study, which employs the Kras^G12D (“K”), Pdx1-Cre (“C”), p53 (“P”) and Rosa^YFP (“Y”) alleles (see Experimental Procedures). Cre-mediated activation of Kras and deletion of one allele of the p53 tumor suppressor is accompanied by recombination of the YFP lineage label. (B) Brightfield and fluorescent images of midgut organs from a CY mouse showing robust and specific fluorescence of the pancreas (outlined); some labeling is also present in the duodenum. (C) Time course of malignant progression in PKCY mice. (D–F) Representative images of malignant progression. Prior to weaning, PKCY mice have histologically normal pancreata (D) but develop PanIN lesions (E) and eventually PDAC (F). (G–I) Images of pancreata from (D–F) stained with an antibody against YFP (green) and N-cadherin (N-cad, red); prior to weaning, scant N-cad staining is seen (G). (J–K) Fluorescent images of lineage-labeled cells derived from the pancreatic epithelium. In Control (CY; Pdx-Cre; YFP) pancreata, YFP⁺ cells express E-cadherin (E-cad; J) but not N-cad (K). Dotted lines indicate YFP⁻ mesenchymal cells. (L–Q) Images of sections from control (CY; L–N) or PDAC (PKCY; O–Q) pancreata co-stained with E-cad and Zeb1 (L, O); YFP and Zeb1 (M, P); and YFP and E-cad (N, Q). Insets and arrowheads in O–Q show hi- magnification views of cells which co-express an epithelial and mesenchymal marker (O), epithelial-derived (YFP⁺) cells which have acquired expression of the mesenchymal marker Zeb1 (P) or epithelial-derived cells which have lost expression of the epithelial marker E-cad (Q). Scale bars denote 100μm in G–I and L–Q and 10μm in J–K. See also Figure S1.

Figure 2. EMT precedes tumor formation

(A–D) In pancreata taken from 8–10 wk-old PKCY mice, EMT is observed in regions of acinarto-ductal metaplasia with inflammation (ADMI; D), PanIN 2 (B), PanIN 3 (C), but not in PanIN 1 lesions (A). Arrows show individual YFP⁺ cells (green) that also express Zeb1 (red). (E) Quantification of observations from A–D, showing the percentage of each type of lesion having at least one cell that has undergone EMT; numbers reflect at least 10 medium-powered fields from each of five PanIN mice. (F) Strategy for isolating YFP⁺ epithelially-derived cells from the pancreas; the purity of the YFP⁺ population was confirmed by a repeat FACS analysis. (G) Transcriptional analysis of sorted YFP⁺ pancreas cells from lineage-labeled CY control (n=4), PanIN (n=6), and PDAC (n=5) pancreata. Bar graph data are presented as mean ± SD in this and subsequent figures. *, p<0.01; **, p<0.001 by two-tailed Student's t-test in this and subsequent figures, unless otherwise noted. Scale bars, 20μm. See also Figure S2.

Figure 3. Hematogenous spread and liver seeding precede tumor formation

(A and B) Images showing individual YFP⁺ cells (green) intermingled with stromal cells prior to tumor formation in a 10 wk-old PKCY PanIN mouse (A). Delaminated YFP⁺ cells have a spindle-shaped morphology and express Zeb1 (boxes i–iii); they are indistinguishable from surrounding Zeb1⁺YFP⁻ stromal cells by H&E staining of an adjacent section (B). (C and D) FACS analysis of blood samples from age-matched CY Control (C) and PKCY PanIN mice (D). YFP fluorescence and a stain for the leukocyte marker CD45 are depicted on the X- and Y-axes of the FACS plot. YFP⁺CD45⁻ cells were seen in the blood of PanIN (D) and PDAC (not shown) animals (boxed area indicates representative gating and absolute number of YFP⁺ cells). (E) Quantification of circulating YFP⁺ pancreatic cells (CPCs). Mean CPC numbers (per ml blood) were 3.65±3.76 (CY control, n=13), 32.8±26.2 (PanIN, n=17), and 97.3±48.9 (PDAC, n=18) (p<0.001). (F–G) Phase-fluorescent images showing epifluorescence of a sorted YFP⁺ cell. (H) Genomic PCR showing the presence of the recombined YFP allele in YFP⁺ cells but not YFP⁻ cells. Pancreatic DNA containing the recombined allele was included as a positive control. (I) Expression of transcripts encoding YFP, Pdx1, and E-cad, comparing sorted YFP⁺ and YFP⁻ cells and measured by qPCR (±SD). (J–K) Sanger sequencing after PCR amplification of cDNA showing that YFP⁺ CPCs express a mutant Kras allele which harbors an altered codon 12 (G→A, highlighted). (L–N) CPCs from 8–10 wk-old PKCY animals seed the liver. (L) Micrometastasis in a liver from a tumor-bearing mouse (“PDAC Liver”). (M and N) Individual CPCs seed the liver at the PanIN stage (“PanIN Liver”); vascular lumens are outlined. Scale bar, 40μm for A–B; 5μm for L–N. See also Figure S3.

Figure 4. CPC characterization

(A) Quantification of CPCs after sampling from the left atrium or ventricle (Left) or right atrium or ventricle (Right) of the same animal (n=3). (B) Quantification of FACS staining for epithelial and mesenchymal markers in CPCs obtained from PanIN or PDAC mice (n=6–8 for each data point). (C and D) Quantification of YFP⁺ cells from the pancreas (C) and circulation (D) in PKCY PanIN and PDAC mice that stained positive for the putative pancreatic cancer stem cell markers CD24 and CD44. (E and F) Quantification of survival (E) or clonal growth (F) of YFP⁺ cells obtained from lineage-labeled Control (CY), PanIN and PDAC mice in ultra-low attachment wells. Bar graphs show the number of wells (out of 96 wells seeded with a single cell) exhibiting any live YFP⁺ cells (E, inset) or evidence of clonal growth (F, inset) after 7d. p-values for paired 2-tailed Student's t-tests are shown. Scale bars, 10μm. See also Figure S4.

Figure 5. Epithelial and mesenchymal states are plastic

(A) Schematic of orthotopic transplantation experiments. (B–E) Fluorescent images taken 3 wks following transplantation of YFP⁺ cells from PDAC mice into NOD/SCID hosts. Tumors form in all mice regardless of E-cad status (n=5 for each condition). YFP⁺E-cad⁺ and YFP⁺E-cad⁻ cells are present in both conditions (C and E), as are YFP⁺Zeb1⁺ and YFP⁺Zeb1⁻ cells (B and D). (F–I) Fluorescent images taken 8 wks following transplantation of YFP⁺ cells from PanIN mice into NOD/SCID hosts. After transplantation of YFP⁺E-cad⁺ cells, no tumors are found (n=6); the few transplanted YFP⁺ cells that remain are Zeb1⁻ and E-cad⁺ (F, G). Transplantation of YFP⁺E-cad⁻ cells results in tumor formation (H, I). Tumors contain both E-cad⁺ and E-cad⁻ cells (I) as well as Zeb1⁺ and Zeb1⁻ cells (H), providing direct evidence for MET.

Figure 6. Inflammation augments EMT and dissemination

(A–F) Fluorescent images of 8 wk-old control Pdx1-Cre; Rosa^YFP (CY; A, D), Kras^G12D; Pdx1-Cre; Rosa^YFP (KCY; B, E), and PanIN (C, F) pancreata 3d after injection with vehicle (A–C) or cerulein (D–F) to induce acute pancreatitis (n=3 for each condition). YFP⁺Zeb1⁺ cells present in PanIN mice or observed following cerulein treatment of CY and KCY mice are shown (C–F, insets). (G) Quantification of CPC number after 3d of treatment with vehicle or cerulein for mice with the indicated genotypes (n=3 for each group; p<0.05 comparing the two groups of KCY mice by Student's t-test; p=0.014 for the effect of cerulein versus control across all groups by two-way ANOVA). (H–M) Images of 10-wk-old control (H, K) and PanIN pancreata (I–J, L–M) following 7d treatment with vehicle (DMSO; H–J) or dexamethasone (Dex; K–M) and analyzed 24h after the last injection. YFP⁺Zeb1⁺ cells are seen in vehicle-treated controls (J, inset) but not in Dex-treated animals (M, inset). (N) Quantification of CPC number in vehicle- and Dex-treated PanIN mice (p=0.029, n=6 for each group). See also Figure S6.