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INTRODUCTION Mouse models are instrumental in advancing our understanding of glioblastoma (GBM), however many commonly used models have features that limit their practicality or applicability. These limitations can include an incomplete immune context, a requirement for complex breeding strategies, high cost of specialized lines, and unpredictability in where tumors arise. We sought to develop and validate a mouse modeling system for some of the major human GBM subtypes that overcame these practical challenges. METHODS In vivo electroporation was used to introduce genetic alterations into periventricular cells of early postnatal C57Bl6/j mice. PiggyBac transposon/transposase and CRISPR-Cas9 systems were used to overexpress (OE) or knock out (KO) genes associated with mesenchymal (Nf1-KO/Pten-KO/p53-KO), classical (EGFRvIII-OE/Cdkn2a-KO/Pten-KO), and proneural (Pdgfra-OE/Cdk4-OE/p53-KO) IDH-WT GBM subtypes. KO or OE was confirmed by Indel Detection by Amplicon Analysis and immunofluorescent staining (IF). Tumours were analyzed for histologic, immunohistochemical, and transcriptional (RNAseq) features. RESULTS Tumors arose with near complete penetrance and median survivals of 36 to 116 days amongst models. All tumors were GFAP-expressing high-grade gliomas, with distinct phenotypes associated with different genetic combinations. Nf1-KO/Pten-KO/p53-KO tumors were composed of spindle cells. EGFRvIII-OE/Cdkn2a-KO/Pten-KO and Pdgfra-OE/Cdk4-OE/p53-KO tumors showed prevalence of small, round cells. Molecularly, Nf1-KO/Pten-KO/p53-KO tumors were enriched for human mesenchymal signature and displayed more differentiated phenotype. The Nf1-KO/Pten-KO/p53-KO model also showed increased stromal cells and macrophages. The other models had mixed proneural and classical profiles and were enriched for OPC- and NPC-like gene signatures found in human GBM. Once tumors formed, cells from each model were transplantable into C57Bl6/j mice to generate subsequent tumors. CONCLUSION We developed and validated a rapid, versatile, and reproducible system to model GBMs. These models allow for controlled study of GBM pathogenesis, progression, and treatment response, and allow for robust generation of syngeneic implantable mouse models that can serve as valuable tools for preclinical testing.

We analyzed the role of WIF1 in normal and acanthotic epidermis of 12-O-tetradecanoylphorbol-13-acetate (TPA) or all-trans-retinoic acid (ATRA)-treated and basal cell carcinoma (BCC)-bearing mice. WIF1 protein is located in the follicular infundibulum and interfollicular epidermis (IFE) in murine back skin. Within the hyperplastic epidermis of TPA- or ATRA-treated or BCC-bearing murine skin, WIF1 and Keratin 10 overlap in Ki67⁻ suprabasal layers, while basal epidermal layers expressing Ki67, and BCCs expressing Wif1 mRNA, are free of WIF1 protein. This is similar in human skin, with the exception that WIF1 protein is found in single Ki67⁻ basal epidermal cells in normal skin and additionally in Ki67+ cells in acanthotic skin. Wif1-deficiency enhances acanthosis of the murine BCC-associated epidermis, which is accompanied by an increase of Ki67+ and of Sca-1+ basal cells. WIF1 overexpression in allografted BCC-derived keratinocytes prevents growth and keratinization, involving enhanced phosphorylation of protein kinase C and extracellular signal–regulated kinase 1 and arguably factors secreted by the in vivo environment. In summary, WIF1 protein marks suprabasal layers in the normal IFE. It is also present in the epidermis overlaying BCCs where it diminishes proliferation of basal cells and production of differentiating suprabasal cells. In addition, WIF1 can prevent proliferation and keratinization of BCC-related keratinocytes.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin tumor in humans. Although current therapies are sufficient to clear the tumor in many cases, the overall risk of cSCC metastasis is still 5%. Alternative treatment options could help to overcome this situation. Here we focused on the role of the Hedgehog (HH) signaling pathway and its interplay with epidermal growth factor receptor (EGFR) signaling in cSCC. The analyses revealed that, despite lack of Sonic HH (SHH) expression, a subset of human cSCC can express GLI1, a marker for active HH signaling, within distinct tumor areas. In contrast, all tumors strongly express EGFR and the hair follicle stem cell marker SOX9 at the highly proliferative tumor-stroma interface, whereas central tumor regions with a more differentiated stratum spinosum cell type lack both EGFR and SOX9 expression. In vitro experiments indicate that activation of EGFR signaling in the human cSCC cell lines SCL-1, MET-1, and MET-4 leads to GLI1 inhibition via the MEK/ERK axis without affecting cellular proliferation. Of note, EGFR activation also inhibits cellular migration of SCL-1 and MET-4 cells. Because proliferation and migration of the cells is also not altered by a GLI1 knockdown, GLI1 is apparently not involved in processes of aggressiveness in established cSCC tumors. In contrast, our data rather suggest a negative correlation between Gli1 expression level and cSCC formation because skin of Ptch+/- mice with slightly elevated Gli1 expression levels is significantly less susceptible to chemically-induced cSCC formation compared to murine wildtype skin. Although not yet formally validated, these data open the possibility that GLI1 (and thus HH signaling) may antagonize cSCC initiation and is not involved in cSCC aggressiveness, at least in a subset of cSCC. Open-Access-Publikationsfonds 2019 peerReviewed

Hedgehog (HH) signaling is known to be essential during the embryonal development of the pituitary gland but the knowledge about its role in the adult pituitary and in associated tumors is sparse. In this report we investigated the effect of excess Hh signaling activation in murine pituitary explants and analyzed the HH signaling status of human adenopituitary lobes and a large cohort of pituitary adenomas. Our data show that excess Hh signaling led to increased proliferation of Sox2+ and Sox9+ adult pituitary stem cells and to elevated expression levels of adrenocorticotropic hormone (Acth), growth hormone (Gh) and prolactin (Prl) in the adult gland. Inhibition of the pathway by cyclopamine reversed these effects indicating that active Hh signaling positively regulates proliferative processes of adult pituitary stem cells and hormone production in the anterior pituitary. Since hormone producing cells of the adenohypophysis as well as ACTH-, GH- and PRL-immunopositive adenomas express SHH and its target GLI1, we furthermore propose that excess HH signaling is involved in the development/maintenance of hormone-producing pituitary adenomas. These findings advance the understanding of physiological hormone regulation and may open new treatment options for pituitary tumors.

Pluripotent stem cells have the therapeutic potential in future regenerative medicine applications. Therefore, it is highly important to understand the molecular mechanisms governing the pluripotency and differentiation potential of these cells. Our current knowledge of pluripotent cells is largely limited owing to the candidate gene/protein approach rather than studying the complex interactions of the proteins. Experimentally, yeast two-hybrid system (Y2H) is by far the most useful and widely used method to detect the protein–protein interactions in high-throughput screenings. Unfortunately, currently there is no GAL4-based pluripotent stem cell-specific cDNA library available for screening the interaction proteins impeding the large-scale studies. In this study, we report the construction of Y2H cDNA libraries derived from mouse pluripotent embryonic stem cells (ESCs) and multipotent adult germ-line stem cells (maGSCs) in GAL4-based Y2H vector system with very high transformation efficiency. Furthermore, we have constructed two different baits and screened for interaction partners in an effort to characterize the libraries and also as a part of our ongoing studies. Consequently, many putative interaction proteins were identified in both cases and their interaction was further validated by direct-Y2H. The observed interactions between bait proteins and their respective analyzed putative interaction proteins were further confirmed using two independent approaches in mammalian cells, thus highlighting the biological significance of the identified interactor (s). Finally, we would like to make these cDNA libraries as a resource that can be distributed to the research community. peerReviewed