PMC

Brg1 ablation sensitizes adult duct cells to Kras-driven initiation of IPMN-like lesions. (a) Tamoxifen administration scheme for experiments using Hnf1b–Cre^ERT2 mice. The tamoxifen treatment resulted in recombination of the Brg1 allele in most pancreatic duct cells as determined by the number of Brg1-negative duct cells in Hnf1b–Cre^ERT2; Kras^G12D; Brg1^f/f mice. (b–m) H&E staining (b–d), Brg1 staining (e–g), Ki67 staining (h–j) and p-MAPK staining (k–m) of the indicated genotypes. Scale bars: 100 µm (b–d) and 50 µm (e–m).

Brg1 ablation abrogates PanIN formation from adult acinar cells. (a) Tamoxifen (TAM) administration scheme for experiments using Ptf1a–Cre^ER mice. (b,c) H&E staining. (d,e) Brg1/Alcian blue staining. (f,g) Alcian blue staining. (h) Quantification of PanIN incidence in Ptf1a– Cre^ER; Kras; Brg1^f/+ (Brg1 het) and Ptf1a–Cre^ER; Kras; Brg1^f/f (Brg1 KO) mice 6 weeks after tamoxifen induction. n = 3 mice per group, 1 entire pancreas section per mouse analysed; values are shown as mean ± s.d., P values were calculated with the unpaired t-test. (i) Quantification of Brg1 positive/negative PanINs in Ptf1a–Cre^ER; Kras; Brg1^f/+ (Brg1 het; total of 97 PanINs examined) and Ptf1a–Cre^ER; Kras; Brg1^f/f (Brg1 KO; total of 30 PanINs examined) mice. Scale bars, 100 µm (b,c), 50 µm (d,e); 500µm (f,g).

Loss of Brg1 cooperates with Kras to form neoplastic cystic lesions. (a,b) Macroscopic view of a Ptf1a–Cre; Kras^G12D; Brg1^f/f pancreas at 9 weeks of age (b shows magnified view of the cysts). The pancreas showed extensive grape-like, multilocular fluid-filled cystic structures throughout, ranging from <1mm up to 5mm in diameter. (c) H&E staining of Ptf1a–Cre; Kras^G12D; Brg1^f/f pancreas at 9 weeks. (d–r) Histological analysis of a Ptf1a–Cre; Kras^G12D; Brg1^f/f (f,i,l,o,r) pancreas at 9 weeks of age compared with age-matched Ptf1a–Cre; Kras^G12D (d,g,j,m,p) and Ptf1a–Cre; Kras^G12D; Brg1^f/⁺ (e,h,k,n,q) pancreata. (d–f) Detailed H&E staining; asterisks mark acinar-to-ductal metaplasia and arrows mark PanINs. Amylase staining (g–i), cytokeratin 19 (CK19) staining (j–l), Alcian blue staining (m–o) and Brg1 staining (p–r) of the indicated genotypes at 9 weeks of age. Scale bars: 2 mm (c); 100 µm (d–f); 250 µm (g–o); 50 µm (p–r).

Loss of Brg1 leads to reduced pancreas size and duct dilations. (a–c) Macroscopic view of a control (a), Ptf1a–Cre; Brg1^f/+ (b) and Ptf1a–Cre; Brg1^f/f (c) pancreas at the age of 3 weeks. (d–i) Microscopic view of a pancreas of control (d,g), Ptf1a–Cre; Brg1^f/+ (e,h) and Ptf1a– Cre; Brg1^f/f (f,i) mice at the age of 3 weeks. (d–f) Brg1 staining of the indicated genotypes at 3 weeks (D, duct; I, encircled islet; D1 marks a duct with retained Brg1 expression; D2 marks a duct with loss of Brg1 expression). (g–i) Haematoxylin and eosin (H&E) staining at 3 weeks of age (arrow in i marks occasional duct dilatation). (j) Relative pancreatic weight (PW) to body weight (BW) ratio of control (n = 6), Ptf1a– Cre; Brg1^f/+ (n = 7), and Ptf1a–Cre; Brg1^f/f (n = 3) pancreas at the age of 3 weeks. Values are shown as mean ± s.e.m.; unpaired t-test was performed to calculate P values. Scale bars: 50µm (d–f) and 250µm (g–i).

Cystic neoplastic lesions resemble human IPMNs but not MCNs. (a,b) To prove a possible connection of the cystic neoplastic lesions to the pancreatic duct system, blue dye (bromophenolblue) was injected in the common bile duct to allow retrograde filling of the duct system. (c) H&E staining shows a connection of a cystic neoplastic lesions in a Ptf1a–Cre; Kras^G12D; Brg1^f/f mouse to the pancreatic duct (arrow). (d–i) ERα (oestrogen receptor) and PR (progesterone receptor) staining of cystic lesions of Ptf1a–Cre; Kras^G12D; Brg1^f/f mice (d,g), Ptf1a–Cre; Kras^G12D mice (e,h) that were treated with caerulein to induce pancreatitis (arrows denote positive cells), and of murine uterus that served as a positive control (f,i). The stroma of the cystic lesions showed very little ERα and PR expression comparable to that found in the negative control. Scale bars: 1 mm (c) and 50 µm (d–i).

Molecular characterization of neoplastic cystic lesions. (a–c) The IPMN-like lesions in Ptf1a–Cre; Kras^G12D; Brg1^f/f mice showed only few apoptotic cells as assessed by cleaved caspase 3 expression similar to PanINs of Ptf1a–Cre; Kras^G12D mice. (d–f) Oncogenic Kras is known to promote activation of the MAP-kinase signalling pathway, and we observed phosphorylated (p-MAPK) in PanINs of Ptf1a–Cre; Kras^G12D mice and in the cystic lesions in Ptf1a–Cre; Kras^G12D; Brg1^f/f mice, whereas no p-MAPK was present in control animal tissue. (g–o) Further expression analysis was performed for Pdx1 (g–i), Hes1 (j–l; arrow in j marks centroacinar cell) and Sox9 (m–o) of the indicated genotypes at 9 weeks of age. Dashed lines show the border of Sox9-positive and -negative epithelial cells. Scale bars: 50 µm (a–o).

IPMN lesions progress to form PDA with short latency but carry a much better prognosis for survival than PanIN-PDAs. (a–d) H&E staining reveals that cysts in Ptf1a–Cre; Kras^G12D; Brg1^f/f mice contain varying degrees of dysplasia ranging from low-grade (a) and intermediate-grade (b) to severe dysplasia (c,d shows a higher magnification; carcinoma in situ). (e–g) Staining of PDA in Ptf1a–Cre; Kras^G12D; Brg1^f/f mice, macroscopic (e) and microscopic (f,g; H&E) view (f: arrow marks adjacent cystic lesion). (h–l) CK19 (h), Brg1/CK19/DAPI co-staining (i; note retained Brg1 expression in non-cancerous stroma cells), Pdx1 (j), phospho-Stat3 (k; p-Stat3) and cleaved caspase 3 staining (l) of Ptf1a–Cre; Kras^G12D; Brg1^f/f cancer cells. (m) Cancer incidence at indicated time points for each genotype. *At 3 weeks one of the Ptf1a–Cre; Kras^G12D; Brg1^f/f mice developed an undifferentiated epithelial cancer not PDA (the tumour was CK19-negative but positive for E-cadherin). All other cancers observed were PDA. (n) Overall survival analysis of the indicated mouse genotypes: Ptf1a–Cre; Kras^G12D (green, n = 26), Ptf1a–Cre; Kras^G12D; Brg1^f/+ (black, n= 48), Ptf1a–Cre; Kras^G12D; Brg1^f/f(red, n = 57) and Ptf1a–Cre; Kras^G12D; p53^f/+ mice (blue, n = 37). Log-rank test revealed a significantly longer survival of Ptf1a–Cre; Kras^G12D; Brg1^f/f compared with Ptf1a–Cre; Kras^G12D; p53^f/+ mice (P<0.0001). Overall median survival of PanIN-PDA mice was 19 weeks, compared with 48 weeks in IPMN–PDA mice. Scale bars: 50 µm (a,b,d,g–l); 250 µm (c); 500µm (f).

IPMN–PDA cells are intrinsically less proliferative than PanIN-PDA cells and carry a distinct molecular profile. (a) Co-staining for pHH3 and CK19 with DAPI of PanIN-PDA (Ptf1a–Cre; Kras^G12D; p53^f/+) and IPMN–PDA (Ptf1a–Cre; Kras^G12D; Brg1^f/f). Arrows indicate pHH3/CK19 double-positive proliferating cancer cells. (b) Percentage of pHH3- or Ki67-positive PDA cells of PanIN-PDA mice (n = 7 for pHH3 and n = 6 for Ki67) and IPMN–PDA mice (n = 8); unpaired t-test was performed to calculate P values. (c) Xenograft model of cancer cell lines derived from Ptf1a–Cre; Kras^G12D; Brg1^f/f (red lines: 2 independent cancer cell lines) compared with Ptf1a–Cre; Kras^G12D; p53^f/+ PDAs (blue lines: 2 independent cancer cell lines), and Ptf1a–Cre; Kras^G12D PDAs (green lines: 2 independent cancer cell lines) in vivo. Calculated doubling times were 5.1 and 5.6 days for Ptf1a–Cre; Kras^G12D; Brg1^f/f versus 3.1 and 3.2 days for Ptf1a–Cre; Kras^G12D; p53^f/+ versus 3.0 and 4.3 days for Ptf1a–Cre; Kras^G12D. For each cell line 8 subcutaneous tumours were quantified; values are shown as mean ± s.e.m. (d) H&E and Brg1 staining of subcutaneous tumours of xenograft model of cancer cell lines derived from Ptf1a–Cre; Kras^G12D; Brg1^f/f and Ptf1a–Cre; Kras^G12D; p53^f/+ PDAs. (e) Depicted are the 50 most downregulated genes identified by RNA deep sequencing analysis in IPMN–PDAs compared with PanIN-PDAs. (f) Chromatin immunoprecipitation analysis of PanIN-PDA cell lines detailing the association of Brg1 with promoter regions of Cdkn2a,Hmga2,Sox9,MMP7,MMP15,Gabrp,Clic3,Adamts1 and Smarca2. Values are expressed as fold enrichment over IgG control. Numbers in parentheses indicate upstream location of the promoter sequence with regard to the transcriptional start site (1×10⁶ cells per chromatin immunoprecipitation; n = 4 independent experiments), values are shown as mean ± s.e.m. Scale bars: 100 µm (a); 50 µm (d).