Dual reporter genetic mouse models of pancreatic cancer identify an epithelial‐to‐mesenchymal transition‐independent metastasis program

• DOI: 10.15252/emmm.201809085
• Publication Date: 2018-08-17T07:34:26Z
• Authors: Yang Chen; Valerie S LeBleu; Julienne L Carstens; Hikaru Sugimoto; Xiaofeng Zheng; Shruti Malasi; Dieter Saur; Raghu Kalluri
• Keywords: 0301 basic medicine; micrometastasis; Medicine (General); Epithelial-Mesenchymal Transition; Integrases; pancreatic ductal adenocarcinoma; dual‐recombinase system; QH426-470; partial epithelial‐to‐mesenchymal transition; Actins; 3. Good health; Pancreatic Neoplasms; Disease Models, Animal; Mice; 03 medical and health sciences; R5-920; Genes, Reporter; Genetics; metastasis; Animals; S100 Calcium-Binding Protein A4; Neoplasm Metastasis; Research Articles

Research Article17 August 2018Open Access Source DataTransparent process Dual reporter genetic mouse models of pancreatic cancer identify an epithelial-to-mesenchymal transition-independent metastasis program Yang Chen Department Cancer Biology, Metastasis Center, University Texas MD Anderson Houston, TX, USA Search for more papers by this author Valerie S LeBleu Julienne L Carstens Hikaru Sugimoto Xiaofeng Zheng Shruti Malasi Dieter Saur Medicine II Klinikum rechts der Isar, Technische Universität München, Germany German Center (DKFZ) and Consortium (DKTK), Heidelberg, Raghu Kalluri Corresponding Author [email protected] orcid.org/0000-0002-2190-547X Information Chen1, LeBleu1, Carstens1, Sugimoto1, Zheng1, Malasi1, Saur2,3 *,1 1Department 2Department 3German *Corresponding author. Tel: [phone]; E-mail: EMBO Mol Med (2018)10:e9085https://doi.org/10.15252/emmm.201809085 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Epithelial-to-mesenchymal transition (EMT) is recognized eukaryotic cell differentiation that also observed in association with invasive tumors. Partial EMT carcinomas imparts cells mesenchymal-like features proposed as essential metastasis. Precise determination frequency partial tumors its functional role metastases needs unraveling. Here, we employed mesenchymal mice driven αSMA-Cre Fsp1-Cre genetically engineered develop spontaneous ductal adenocarcinoma (PDAC) monitor program. Both αSMA- Fsp1-Cre-mediated programs were primary The established primarily composed without evidence program, assessed our fate mapping approach. In contrast, metastatic exhibiting restricted isolated single or micrometastases (3–5 cells). Collectively, studies large nodules preserved epithelial phenotype potentially unravel novel PDAC. Synopsis considered This study identifies non-EMT-mediated lineage tracing via dual-recombinase system fluorescence-switching reporters transgenes. 2–3% PDAC tumors, captured lineage-tracing system. Established lung liver determined acquisition markers such αSMA, FSP1 vimentin. Metastatic only disseminated cells), but did not grow become secondary Introduction (EMT), conversion phenotype, associated loss apical–basal polarity morphology (Hay, 1995; Thiery, 2002; Weinberg, 2009). Previous have collectively offered detection circulating tumor (Rhim et al, 2012; Yu 2013; Javaid 2015) conducted gain-of-function/loss-of-function assays targeting EMT-inducing transcription factors Twist, Snail, Slug, Zeb1 (Guaita Arumugam 2009; Wellner Taube 2010; Tsai 2012). These gave mechanistic insights into molecular basis linked context cancer, revealed fibroblast-specific protein-1 (Fsp1, called S100A4), Zeb1, Snail-expressing cells, defined early formation Furthermore, > 20% macrometastases expression marker, Fsp1 (Aiello 2016). Using similar (GEMMs) (PDAC), previously reported deletion Snai1 (Snail) Twist1 (Twist) was dispensable reduced chemoresistance (Zheng 2015). breast although burden (Tran 2014), over-expression miR-200 (which targets Zeb2 transcriptional repression E-cadherin) reduce despite impact on (Fischer To capture multiple been used immunolabeling lineage-tagged which included α-smooth muscle actin (αSMA), (S100A4), vimentin (Trimboli 2008; Rhim Fischer 2015; Aiello 2016; Zhao KPC (LSL-KrasG12D/+;Trp53R172H/+;Pdx1-Cre) GEMM (Hingorani 2005), combined conventional loxP-STOP-loxP-Reporter transgene, has their While approach strictly allows labeling based Cre-loxP system, identification reporter-labeled relies antibody-based tissue section may accurately evaluated directly facilitated dual GEMMs. colleagues next-generation (DRS) integrating both Flippase (Flp)-FRT (Schonhuber 2014). With distinct genes are independently manipulated under control Cre Flp recombinases, respectively. FSF-KrasG12D/+;Trp53frt/+;Pdx1-Flp (KPF) GEMMs exhibit analogous progression when compared establish transgenic strains using DRS achieve dynamic dual-fluorescence transition, monitoring cells. Keeping mind (Fsp1) (αSMA) (Kalluri Wang Li 2016), bred KPF strain dual-fluorescence-switchable reporter, R26Dual (Rosa26-CAG-loxP-frt-Stop-frt-FireflyLuc-EGFP-loxP-RenillaLuc-tdTomato). strategy Pdx1-lineage express EGFP, while activate αSMA promoters positive tdTomato. Importantly, EGFP+ upon Fsp1, irreversibly lose EGFP gain tdTomato expression. Therefore, during entire course lifespan, acquired will remain tdTomato+/EGFP− even if they subsequently gene expression, reverting speculated mesenchymal-to-epithelial (MET) niches. design enables Fsp1-associated metastases. Results Characterization fluorescence KPF;αSMA-Cre;R26Dual We first generated (FSF-KrasG12D/+;Trp53frt/+;Pdx1-Flp);αSMA-Cre;R26Dual (Rosa26-CAG-loxP-frt-Stop-frt-FireflyLuc-EGFP-loxP-RenillaLuc-tdTomato) mice. use marker transgene motivated previous study, wherein analyses conditional Snail Twist significant measured Zeb2, Slug Notably, these findings, KPC;YFP (LSL-KrasG12D/+;Trp53R172H/+;Pdx1-Cre;R26LSL-YFP) employing lineage-traced 2015), validated additional (Appendix Fig S1). percent expressing findings anti-αSMA antibodies, antibodies αSMA-expressing could be detected Figs S2 S3). current mice, induces oncogenic KrasG12D concurrent heterozygous p53 Pdx1-Flp-expressing Flp-FRT-based alleles induced PDAC, exhibited model (Fig 1A–C). PanIN, lesions prominent cytokeratin-19 (CK19) 1B C, Appendix αSMA-positive myofibroblasts 1D). include (with sequence permanently removed mechanism) EGFP-to-tdTomato captures αSMA-related switch ensures once if/when revert morphology, possibly (MET). Figure 1. Dual-recombinase A. Genetic induce Pdx1-Flp-FRT recombination (DRS). B. Representative sections PanIN (stage 1-3) stained hematoxylin eosin (H&E) immunohistochemistry. panels depicted S3. C. H&E-stained CK19-immunostained (arrows) D. Pdx1-Flp (either cells) Rosa26-CAG-loxP-frt-Stop-frt-FireflyLuc-EGFP-loxP-RenillaLuc-tdTomato (R26Dual) tracer. Download figure PowerPoint 2A B). Pancreatic Pdx1-Flp-induced positivity majority expressed CK19. (CK19-negative) desmoplasia B, S4A All areas (non-necrotic) examined indiscriminately peri-tumoral intra-tumoral areas. Nearly all CK19+ report-driven (> 95%, 2B). A consistent proportion (~1.8% per visual fields) (tdTomato+/CK19+), supporting αSMA-associated launch confirm keeping heterogeneous levels CK19, carried out experiments E-cadherin Similar results obtained, EGFP-expressing S4C). discrete (0.5%) EGFP/tdTomato double-positive diminishing emerging expression) 2B), reflecting retained proteins start at onset αSMA-Cre-driven phenotype. 2. reveal trace images from intrinsic signals, combination CK19 immunofluorescence co-staining. Arrow indicates tdTomato+CK19+ tumor. Quantification percentage EGFP-positive, tdTomato-positive, (3 fields mouse, n = 3 mice; presented mean ± SEM). (circled area) Magnified frame shows histology part metastasis, subsequent staining. circled area cell. data available online figure. Data 2 [emmm201809085-sup-0002-SDataFig2.xlsx] evaluating 20 different each mouse. any tdTomato+ (Table 1). containing than 10 1, 2C D), others (Bailey-Downs within parenchyma and/or (small clusters about 3-5 D). Macrometastases shown lack IHC visualized ex vivo imaging S5A). Further, specificity αSMA-Cre-induced pattern co-localized antibody-mediated 3A Of note, noted co-localize Fsp1/S100A4 3C), another important identifying Neilson, 2003; suggest metastases, (CK19+, no αSMA-Cre-captured program), emerges (CK19+ program). Table Summary experimental Strain name KPF;αSMA-Cre;R26mT/mG KPF;Fsp1-Cre;R26Dual Mouse ID J37 J317 J488 J402 A762 A369 219x 261x DOB 15-04-2015 24-06-2015 05-08-2015 13-07-2015 08-07-2016 18-05-2016 20-09-2016 DOD 20-08-2015 11-09-2015 11-06-2016 13-12-2015 22-10-2016 20-08-2016 15-12-2016 23-01-2017 Age (day) 127 79 311 153 106 94 86 125 Moribund Y Body weight (g) NR 20.48 37.46 17.21 21.5 17.1 Tumor 0.76 1.83 2.04 0.82 0.74 Gender M F Total Lung Mets Non-EMT/Macro 2/8 – EMT/Macro 0/8 Non-EMT/micro EMT/micro 3/8 Liver 5/8 1/8 7/8 ID, identification; DOB, date birth; DOD, death; g, gram; M, male; F, female; Y, yes; NR, recorded; –, observed. 3. Lineage A, colocalization (as indicated arrows) between αSMA-induced staining (A) (B) tdTomato+Fsp1+ Fidelity lines Based flow cytometry further confirmed fluorescent microscopy 4A). TGF-β treatment morphological change affirming activity 4B). upregulated transcript Fsp1/S100A4, (Acta2), fibronectin (FN1), (Snai1), (Twist1), type I collagen α1 (Col1a1), suppressing (Cdh1) (Krt19) level 4C). Although down-regulated vitro treatment, still highly easily detectable S5B), enabled sites. 4. induction Schematic isolation tissues FACS. differential interference contrast (DIC) microscopic treated (5 ng/ml, 96 h) EMT. Relative 48 h), 4 independent (results SEM; Krt19: **P 0.0007, Cdh1: 0.0041, Vim: P 0.6033, S100a4: 0.0082, Acta2: ***P 0.0003, Fn1: 0.0004, Col1a1: *P 0.0424, Twist1: 0.0187, Snai1: 0.0003). Significance paired, two-tailed t-test. NS, significant. [emmm201809085-sup-0003-SDataFig4.xlsx] Dual-fluorescence confirms non-EMT program-associated validate linage next (Rosa26-CAG-loxP-tdTomato-loxP-EGFP) 5A, S6A), reverse "green-to-red" earlier (αSMA-expressing staining) documented 5B, S6B), obtained 2). exclusively ubiquitous 5C, Non-EMT S6C). (colonies 3–5 5C model, harboring extensively utilized R26mT/mG provided confirmation findings. 5. Examination Rosa26-CAG-loxP-tdTomato-loxP-EGFP (R26mT/mG) (Met; C) Arrows indicate EGFP+CK19+ (Met) Fsp1-Cre-captured It argued most predominant order address thesis, mesenchymal-specific promoter drives Cre-recombinase 6A, S7A). initiate generating Consistent S1) found small portion (~2.7%) 6B C). (maintained phenotype) 6D). either (established macrometastases) 1), tumor, Fsp1/tdTomato-positive other vimentin, 6E, S7B–D). fibroblasts stroma minimal overlap fibroblast subpopulation S8A). Such simultaneous antibody cryosections KPF;Cre-negative;R26Dual S8B). efficiency robust Fsp1-Cre-induced S8C). labeled staining, Cre-loxP-based 6. Indication Fsp1-driven Fsp1-expressing E. areas) Fsp1-induced 6 [emmm201809085-sup-0004-SDataFig6.xlsx] Discussion contribution dissemination remains largely unknown extensive investigation. due transient nature