Evidence of renal angiomyolipoma neoplastic stem cells arising from renal epithelial cells

Renal angiomyolipomas (AML) contain an admixture of clonal tumour cells with features of several different mesenchymal lineages, implying the existence of an unidentified AML neoplastic stem cell. Biallelic inactivation of TSC2 or TSC1 is believed to represent the driving event in these tumours. Here we show that TSC2 knockdown transforms senescence-resistant cultured mouse and human renal epithelial cells into neoplastic stem cells that serially propagate renal AML-like tumours in mice. mTOR inhibitory therapy of mouse AML allografts mimics the clinical responses of human renal AMLs. Deletion of Tsc1 in mouse renal epithelia causes differentiation in vivo into cells expressing characteristic AML markers. Human renal AML and a renal AML cell line express proximal tubule markers. We describe the first mouse models of renal AML and provide evidence that these mesenchymal tumours originate from renal proximal tubule epithelial cells, uncovering an unexpected pathological differentiation plasticity of the proximal tubule

Mouse genetic background influences whether expression plus knockdown causes angiosarcoma or undifferentiated pleomorphic sarcoma

Soft tissue sarcomas are rare mesenchymal tumours accounting for 1% of adult malignancies and are fatal in approximately one third of patients. Two of the most aggressive and lethal forms of soft tissue sarcomas are angiosarcomas and undifferentiated pleomorphic sarcomas (UPS). To examine sarcoma-relevant molecular pathways, we employed a lentiviral gene regulatory system to attempt to generate models that reflect common molecular alterations of human angiosarcoma and UPS. Mice were intraveneously injected with MuLE lentiviruses expressing combinations of shRNA against , , and with or without expression of , or . The systemic injection of an ecotropic lentivirus expressing oncogenic together with the knockdown of or was sufficient to initiate angiosarcoma and/or UPS development, providing a flexible system to generate autochthonous mouse models of these diseases. Unexpectedly, different mouse strains developed different types of sarcoma in response to identical genetic drivers, implicating genetic background as a contributor to the genesis and spectrum of sarcomas

Stromal cells are preferentially transduced by lentiviruses following <i>in vivo</i> injection.

<p>(<b>A</b>) tdTomato fluorescence signal in the uterus following intrauterine injection of ecotropic virus expressing Cre. (<b>B</b>) Intrauterine injection of ROSA26-lox-STOP-lox-tdTomato mice with Cre-expressing lenti- and adenoviruses. Arrowheads indicate infected epithelial cells. Scale bar: 100 μm. CTRL indicates non-injected animals.</p

Oncogenic <i>Hras^G12V</i> expression plus knockdown of <i>Cdkn2a</i> using ecotropic lentiviral vectors induces high-grade endometrial stromal sarcoma

<div><p>The uterine corpus represents the most common site for tumour development in the female genital system. Uterine neoplasms are categorised as epithelial, mesenchymal, mixed epithelial-mesenchymal or trophoblastic tumours. In this study we employed a mouse genetic approach using the MuLE lentiviral gene regulatory system to functionally test the ability of ecotropic lentiviruses to model epithelial and mesenchymal uterine malignancies <i>ex vivo</i> and <i>in vivo</i>. We discovered that MuLE lentiviruses efficiently infect uterine stromal cells but not endometrial epithelial cells when injected into the uterus of cycling, pseudopregnant or ovarectomized mice. Consistent with this cellular infection spectrum, we show that intra-uterine injection of ecotropic MuLE viruses expressing oncogenic <i>Hras</i>^<i>G12V</i> together with knockdown of <i>Cdkn2a</i> induce high-grade endometrial stromal sarcomas. These findings establish this approach as an efficient method of generating autochthonous mouse models of uterine sarcomas and in general for performing genetic manipulations of uterine stromal cells <i>in vivo</i>.</p></div

<i>Hras</i>^<i>G12V</i> expression plus knockdown of <i>Cdkn2a</i> causes high-grade endometrial stromal sarcomas.

<p>(<b>A</b>) Schematic of MuLE vectors simultaneously expressing a combination of shRNA against <i>Cdkn2a</i> plus expression of <i>Hras</i>^<i>G12V</i> or expressing shRNAs against <i>Trp53</i> or <i>Pten</i> or a non-silencing (n.s.) control shRNA. All vectors also expressed Luciferase. Numbers of mice that were injected with each vector are shown in the figure. (<b>B</b>) Quantification (mean ± SD) of luciferase signal over time. † Sacrifice of all mice in this group by this time point. (<b>C</b>) Bioluminescence imaging in two mice 7, 22, 28, 34 and 42 days after the injection of MuLE lentiviruses expressing shRNA against <i>Cdkn2a</i> together with <i>Hras</i>^<i>G12V</i> into the uterus of 6-8-week-old SCID/beige mice. Injected mice developed tumours (n = 4) with 80% penetrance. Median overall survival was 49 days. (<b>D</b>) Bioluminescence imaging and photographs of tumour-bearing uterus at the time of sacrifice (day 56 after injection). (<b>E</b>-<b>M</b>) H&E and immunohistochemical stainings of tumours from two different mice using the indicated antibodies. Low magnification scale bar: 1 mm and high magnification scale bar: 100 μm.</p

Establishment of a primary endometrial epithelial cell culturing system.

<p>(<b>A</b>) pEECs stain positively for the epithelial cell markers E-CADHERIN, ß-CATENIN and CYTOKERATIN-7 and negatively for the stromal cell marker VIMENTIN. Bright field image (10x) scale bar: 50 μm, low magnification (20x) scale bar: 100 μm and high magnification (63x) scale bar: 100 μm. (<b>B</b>) Supplementing BIE with 0.5% FCS promotes growth. BIE: basal medium supplemented with insulin, EGF, transferrin und sodium selenite. Scale bar: 50 μm. (<b>C</b>) Adeno-Cre-treated pEECs from <i>Trp53</i>^fl/fl mice and pEECs infected with a lentivirus expressing a miR-30-based shRNA against <i>Trp53</i> were immortalised and could be expanded but lost epithelial morphology. Scale bar: 50 μm.</p

Lentiviral transduction of cultured uterine cells strongly favors stromal cells.

<p>(<b>A</b>) Schematic diagram of Pax8-rtTA-mediated doxycycline-controlled Cre-expression in Pax8-rtTA; LC1; ROSA26-lox-stop-lox tdTomato mice. (<b>B</b>) Doxycycline activates Cre-mediated recombination in glandular und luminar epithelial cells in the endometrium shown by whole organ imaging (left panel) and imaging of frozen histological sections (right panels). Scale bar: 100 μm. (<b>C</b>) Bright field and fluorescence images 1, 4, 6, 11 and 25 days after the isolation of pEECs from the uteri of doxycycline-treated Pax8-rtTA; LC1; ROSA26-lox-stop-lox tdTomato mice. pEECs were infected on day 1 with ecotropic lentiviruses expressing either GFP or sh<i>Cdkn2a</i> plus <i>Hras</i>^<i>G12V</i> and Luciferase. Scale bar: 50 μm. (<b>D</b>) Ability of sh<i>Cdkn2a</i> and <i>Hras</i>^<i>G12V</i>-transduced cells to form tumours within 47 days in allograft experiments (visualised using luciferase imaging in the left panel). Right panels show H&E and immunohistochemical stainings using the indicated antibodies. Low magnification scale bar: 1 cm and high magnification scale bar: 100 μm.</p