Long-term observation reveals high-frequency engraftment of human acute myeloid leukemia in immunodeficient mice

Repopulation of immunodeficient mice remains the primary method for functional assessment of human acute myeloid leukemia. Published data report engraftment in ~40-66% of cases, mostly of intermediate- or poor-risk subtypes. Here we report that extending follow-up beyond the standard analysis endpoints of 10 to 16 weeks after transplantation permitted leukemic engraftment from nearly every case of xenotransplanted acute myeloid leukemia (18/19, ~95%). Xenogeneic leukemic cells showed conserved immune pheno-types and genetic signatures when compared to corresponding pre-transplant cells and, furthermore, were able to induce leukemia in re-transplantation assays. Importantly, bone marrow biopsies taken at standardized time points failed to detect leukemic cells in 11/18 of cases that later showed robust engraftment (61%, termed "long-latency engrafters"), indicating that leukemic cells can persist over months at undetectable levels without losing disease-initiating properties. Cells from favorable-risk leukemia subtypes required longer to become detectable in NOD/SCID/IL2Rγ; null; mice (27.5±9.4 weeks) than did cells from intermediate-risk (21.9±9.4 weeks,; P; <0.01) or adverse-risk (17±7.6 weeks;; P; <0.0001) subtypes, explaining why the engraftment of the first was missed with previous protocols. Mechanistically, leukemic cells engrafting after a prolonged latency showed inferior homing to the bone marrow. Finally, we applied our model to favorable-risk acute myeloid leukemia with inv(16); here, we showed that CD34; +; (but not CD34; -; ) blasts induced robust, long-latency engraftment and expressed enhanced levels of stem cell genes. In conclusion, we provide a model that allows; in vivo; mouse studies with a wide range of molecular subtypes of acute myeloid leukemia subtypes which were previously considered not able to engraft, thus enabling novel insights into leukemogenesis

What influences the decision to drop out? Longitudinal analyses of the interplay between study satisfaction, content knowledge and dropout intention in chemistry, engineering and social sciences

Die Untersuchung der Gründe für den Studienabbruch von Studierenden in MINT-Fächern ist wegen des akuten Fachkräftemangels von großer Bedeutung. Dabei ist bisher wenig über Unterschiede zwischen MINT-Studiengängen, aber auch im Vergleich zu anderen Studiengängen, hinsichtlich der Abbruchgründe bekannt. Die längsschnittlich angelegte Studie untersucht daher individuelle Einflussfaktoren auf den Studienerfolg und Studienabbruch in zwei typischen MINT-Studiengängen (Chemie und Maschinenbau) und kontrastierend dazu in Studiengängen aus dem sozialwissenschaftlichen Bereich (Politikwissenschaft, Soziologie, Soziale Arbeit). Während in bisherigen Studien (z.B. Heublein et al., 2017) fachübergreifende Prädiktoren für den Studienabbruch identifiziert werden konnten, wurde in den Analysen dieser Studie festgestellt, dass sich diese zwischen den Fächern und auch zwischen unterschiedlichen Hochschultypen sehr unterschiedlich auswirken. Vor allem die Sozialwissenschaften zeigen im Vergleich zu den MINT-Fächern einen deutlich geringeren Einfluss des Fachwissens auf den Studienabbruch. (DIPF/Orig.)In view of the current shortage of qualified personnel in STEM subjects, the investigation of the reasons for dropping out of university studies is of great importance. Little is known about the differences between STEM study programmes and other study programmes with regard to the reasons for dropping out. The longitudinal study of this project therefore examines the factors influencing the success and termination of studies in two typical STEM programmes (chemistry and mechanical engineering) and, in contrast, in programmes of the social sciences (political science, sociology, social work). Based on Heublein et al. (2017), data were collected to identify subject-specific factors influencing the dropout of students. The results of path analyses indicate that dropout by students of social sciences is less influenced by content knowledge compared to STEM study programmes. (DIPF/Orig.

Investigation of mechanisms regulating leukemogenesis using mouse xenograft models of human acute myeloid leukemia

Acute myeloid leukemia (AML) is a malignant neoplasia of the blood system and can occur in people at all ages but more frequently affects people older than 65 years. Although the outlook for patients with AML has improved over the past decades, still more than half of young adult and about 90% of elderly patients die from their disease. The main obstacles to cure are refractoriness to initial induction treatment and, more frequently, relapse after apparent remission. From a cellular perspective, relapse is thought to occur from rare cell populations of so-called “leukemia stem cells” (LSCs) which share molecular and functional features with their healthy counterparts, the hematopoietic stem cells (HSCs). Because they underlie complex regulatory mechanisms, particularly also involving other cell types and niches, LSCs need to be studied in vivo. For human LSCs, such studies are confined to xenotransplantation models, which usually are performed in immunosuppressed mice. In this thesis, a better understanding of human AML was built up by (1) establishing and improving a stable xenotransplantation model, (2) by identifying a novel LSC marker based on the concept of immune escape and (3) by unraveling novel non-cell autonomous pro-leukemogenic mechanisms involving BM niche and healthy hematopoietic stem and progenitor cells (HSPCs) modulation by CDX2 expressing leukemic cells. Lastly, we use an ex vivo 3-dimensional niche surrogate for expansion of healthy HSPCs. Firstly, an improved transplantation protocol of human AML cells into immunosuppressed NOD/SCID/IL2Rγnull (NSG) mice that mimics the clinical course of the disease in patients was established. In this model, the latency of AML cell engraftment in mice depends on molecular risk groups established in patients and xenogeneic leukemic cells show conserved genetic and phenotypic features. Most importantly, the model enables the engraftment of favorable risk AML subtypes previously considered non-engraftable in NSG mice, opening up new perspectives for in vivo studies on these disease subtypes. We further optimized this model by observing that transplantation at night or under enhanced catecholamine activity favors homing and adhesion of leukemic cells to BM niches, thereby shortening time-to-leukemia in vivo. Next, this model was used for mechanistic in vivo studies on leukemia initiation. In close collaboration with the immunology research group led by Prof. Helmut Salih (University of Tuebingen, Germany), NKG2D-associated im-mune privilege was identified as a feature of human AML LSCs. These findings offer a novel method for LSC isolation in several subtypes of AML and demonstrate in functional assays an unrecognized and targetable mechanism for sensitizing LSCs to immune control. Furthermore, our studies on the transcription factor CDX2, which is expressed in >80% of AML but not detectable in healthy HSPCs, unraveled a novel non-cell-autonomous role by which CDX2 promotes leukemogenesis, namely via DKK1 secretion to outcompete resident healthy HSPCs, which indicates treatment with WNT agonists as potential strategy to treat incipient AML (e.g. AML at minimal residual disease stage). Last, as part of a collaborative project with the research group led by Prof. Ivan Martin (Department of Biomedicine, University of Basel), an ex vivo BM niche surrogate system was analyzed for its potential to expand and analyze healthy cord-blood derived HSPCs in vitro, which could also be adapted to recapitulate pathological situations as leukemia. This could represent a pow-erful tool with wide range of applications, from the identification of factors deregulating niche or blood functions, to the screening of drugs to predict pa-tient-specific response to defined treatments without using xenotransplanta-tion models

In-vitro Untersuchungen zur Amphotericin B-induzierten Hypokaliämischen Periodischen Paralyse

Familial hypokalemic periodic paralysis is a rare inherited muscle disease characterised by episodic attacks of flaccid muscle paralysis. These episodes go along with an increased shift of K+ ions into muscle, what results in hypokalemia. There is also a depolarization of muscle fibers to membrane potentials at which voltage-gated channels are inactivated. In the end, this results in inexcitability and the characteristic paralysis. It is caused by mutations in the voltage-sensing domain of voltage-gated sodium or calcium channels of skeletal muscle. It is supposed that these mutations create a cation leak, which might be the primary pathogenetic cause of the disease. Acetazolamide (AZ), a carboanhydrase-inhibitor, seems to be an effective therapy. In reference to this guess, we conducted an in-vitro study on rat skeletal muscle preparations that we made leaky for cations by Amphothericin B (AMB) and measured the membrane potential with glass microelectrodes. AMB is an ionophore clinically used as an antifungal drug and is well known for its serious side effects. The study showed that there are two populations of fibers with regard to the resting membrane potential. One is polarized close to the Nernst potential of K+, the other is depolarized to about - 60 mV. In low [K+], the fraction of depolarized fibers is larger. The leak induced by AMB leads to the result that there are more depolarized fibers at intermediate [K+] compared to control. Studies with AZ showed that in low [K+] the fraction of polarized fibers is increased about thirty percent. The conclusion is that a depolarizing leak might shift the region of bistability of resting membrane potentials to near physiologic [K+]. Our study shows that AMB increases the fraction of depolarized muscle fibers at physiologic and reduced [K+]. Thus, AMB might be a suitable pharmacological model for hypokalemic periodic paralysis and AZ a stabilizer for the membrane potential

Evaluation of stem cell properties in human ovarian carcinoma cells using multi and single cell-based spheres assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called "cancer stem cells" (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts

Stress and catecholamines modulate the bone marrow microenvironment to promote tumorigenesis

High vascularization and locally secreted factors make the bone marrow (BM) microenvironment particularly hospitable for tumor cells and bones to a preferred metastatic site for disseminated cancer cells of different origins. Cancer cell homing and proliferation in the BM are amongst other regulated by complex interactions with BM niche cells (e.g. osteoblasts, endothelial cells and mesenchymal stromal cells (MSCs)), resident hematopoietic stem and progenitor cells (HSPCs) and pro-angiogenic cytokines leading to enhanced BM microvessel densities during malignant progression. Stress and catecholamine neurotransmitters released in response to activation of the sympathetic nervous system (SNS) reportedly modulate various BM cells and may thereby influence cancer progression. Here we review the role of catecholamines during tumorigenesis with particular focus on pro-tumorigenic effects mediated by the BM niche

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC