Extended Data Figure 2 |. Isolation and functional characterization of epithelial and mesenchymal clones from primary tumours in a conditional reporter GEMM of PDAC.

a, Schematic model of the GEMM. Kras^G12DLSL/+-Tp53^LoxP/LoxP-Pdx1-Cre (KPC^Δ/Δ) mice were crossed with a strain expressing a lineage-tracing fluorescent reporter (R26^mTmG) and a strain expressing a Cdh1^Cfp reporter, in which CFP (cyan fluorescent protein) is expressed as a fusion protein with endogenous E-cadherin to generate the KPC^Δ/Δ-R26^mTmG/+-Cdh1^Cfp/+ dual-reporter model of PDAC. This system allows the isolation of GFP-positive malignant cells and the separation of CFP^high (epithelial) from CFP^low (mesenchymal) sub-populations. b, FACS experiment showing the distribution of CFP^high and CFP^low sub-populations in both the GFP⁺ (tumour cells) compartment and the TdTomato⁺ (stromal) compartment. The reporter shows an absence of CFP⁺ cells in the stromal (TdTomato⁺) compartment and a spectrum of sub-populations in the tumour (GFP⁺) compartment. c, Western blot analysis of the expression levels of SMARCB1 in the GFP⁺CFP^high and GFP⁺CFP^low sub-populations. Vinculin was used as loading control. d, Representative sections showing the levels of SMARCB1, CDH1 and phospho-ERK1/2 in orthotopic transplants of malignant sub-populations, isolated as described above. MS-L-derived transplants were used as controls. e, In vivo characterization of the PDAC sub-populations in the KPC^Δ/Δ-R26^mTmG/+ model of PDAC. Immunofluorescence staining for GFP, SMARCB1, phospho-ERK1/2 and nestin in PDAC originated in the KPC^Δ/Δ-R26^mTmG/+ background strain. Low levels of SMARCB1 and phospho-ERK1/2 and high levels of nestin are a hallmark of the sub-population of invasive GFP⁺ cells. Scale bars: 100 μm (d; e, bottom nine panels), 20 μm (e, top four panels). For gel source data, see Supplementary Figures.