Mendelian transmission of Rosa26-targted CAGs-rtTA3 transgenes.

<p>*Numbers represent: observed (expected) from heterzygote x wild-type crosses.</p

GFP induction and mKate2 expression is uniform in most organs of <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice.

<p>Immunofluorescence stains for GFP and mKate2 in the small intestine and pancreas of ‘no rtTA’, <i>R26-rtTA</i>, <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice following 1 week of doxycycline treatment. All rtTA strains show strong GFP induction in small intestine (<b>A</b>), but only <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> show robust and uniform GFP expression (and mKate2 for <i>RIK</i>) in the pancreatic acinar tissue (<b>B</b>).</p

CAGs-rtTA3 and CAGs-RIK show strong expression in adult tissues.

<p>Whole mount epifluorescence images of small intestine, skin, pancreas kidney and liver from <i>R26-rtTA</i>, <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> transgenic animals (all containing <i>TG-Ren.713</i>). <i>R26-rtTA</i> shows strong expression in intestine and skin but weak or patchy expression in most other solid organs. <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> show almost identical expression patterns in adult mice. <i>CAGs-RIK</i> mice show strong and consistent expression of mKate2.</p

<i>CAGs-LSL-RIK</i> enables tissue-restricted expression of <i>TRE</i>-transgenes in transgenic models of disease.

<p><b>A</b>. Whole mount epifluorescence (top panel) and immunofluorescence images from a quadruple transgenic (<i>CAGs-LSL-RIK;TG-Ren.713;LSL-Kras^G12D;Pdx1-Cre</i>) animal, showing induction of GFP and mKate2 in both normal acinar tissue and pre-neoplastic, Kras^G12D-induced PanIN lesions (top arrow). As observed in AdenoCre treated lungs, some PanIN lesions did not show GFP or mKate2 staining suggesting incomplete LSL excision in a small proportion of cells. <b>B</b>. Immunofluorescent stains for GFP and mKate2 in mammary tissue of <i>CAGs-LSL-RIK;TG-Ren.713;MMTV-Neu;WAP-Cre</i> transgenic mice treated with dox.</p

Adenoviral Cre induces mosaic activation of rtTA and GFP induction in <i>CAGs-LSL-rtTA3</i> and <i>CAGs-LSL-RIK</i> animals.

<p><b>A</b>. Immunofluorescent stains for GFP and mKate2 in liver sections of <i>TG-Ren.713;CAGs-LSL-rtTA3</i> and <i>TG-Ren.713;CAGs-LSL-RIK</i> mice 1 week following intravenous injection of Adenoviral Cre (5×10⁸ PFU) or PBS (<i>CAGs-LSL-RIK</i> only – left panel) and dox treatment. Double transgenic mice exposed to AdenoCre show mosaic expression of GFP (<i>CAGs-LSL-rtTA3</i>) or GFP and mKate2 (<i>CAGs-LSL-RIK</i>). No GFP of mKate2 expression was observed in animals not exposed to Cre. <b>B</b>. Immunofluorescent stains for GFP and mKate2 in lung sections of triple transgenic mice (<i>CAGs-LSL-rtTA3 or RIK;TG-Ren.713;LSL-Kras^G12D</i>). Kras^G12D-induced lung adenomas show strong expression of GFP and mKate2. Lowe panel: higher magnification of the lesion. White arrows indicate rare cells that show mKate2, but not GFP expression.</p

Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.We thank D. L. Bajor (Vonderheide laboratory, University of Pennsylvania) for the gift of the R6560B cells. We thank L. Bojmar for carefully reviewing the paper. We thank S. Rudchenko and M. Barbu-Stevanovic at the Hospital for Special Surgery Fannie E. Rippel Foundation Flow Cytometry Core Facility for expert flow cytometry. We are supported by grants from the Children’s Cancer and Blood Foundation (H.P., D.L.), Manning Foundation (D.L.), Hartwell Foundation (D.L.), Champalimaud Foundation (D.L.), Fundacao para a Ciencia e a Tecnologia (D.L.), Nancy C and Daniel P Paduano Foundation (H.P., D.L.), Mary Kay Foundation (D.L.), Pediatric Oncology Experimental Therapeutic Investigator Consortium (D.L.), James Paduano Foundation (D.L., H.P.), Melanoma Research Alliance (H.P.), Sohn Conference Foundation (H.P.), Beth Tortolani Foundation (D.L., J.B.), Malcolm Hewitt Weiner Foundation (D.L.), Jose Carreras Leukemia Foundation (B.K.T.), Theodore Rapp Foundation (D.L.), American Hellenic Educational Progressive Association 5th District Cancer Research Foundation (D.L.), Charles and Marjorie Holloway Foundation (J.B.), Sussman Family Fund (J.B.), Lerner Foundation (J.B.), Breast Cancer Alliance (J.B.), and Manhasset Women’s Coalition Against Breast Cancer (J.B.).S