Patients' acute lymphoblastic leukemia cells show heterogeneous growth behavior and drug sensitivity in vivo

Acute leukemias consist of heterogeneous cell populations and the most aggressive subpopulation determines prognosis and outcome in each patient. A better understanding of challenging subclones is intensively desired, regarding both genotype and functional phenotype. New therapies are required which eradicate aggressive subpopulations in order to improve the prognosis and cure rate of patients with cancer. Here, we aimed at characterizing single cell clones in order to find putative therapeutic targets. Primary tumor cells from a girl with acute lymphoblastic leukemia (ALL) at first relapse were transplanted into severely immunocompromised mice and lentivirally modified to express the fluorochromes red, green and blue at different amounts and combinations (RGB marking, (Weber et al., 2011)). Single cell clones were generated by limiting dilution transplantation and their uniqueness was verified by LM-PCR. In order to identify challenging subclones, molecularly marked clone mixtures were transplanted into the same recipient mouse to perform competitive in vivo proliferation and drug sensitivity assays and analyzed separately by flow cytometry using their unique expression of molecular markers. In clone mixtures, certain clones were overgrown by others indicating unfavorable slow proliferation. When two clones were mixed and transplanted in groups of mice and animals were treated with glucocorticoids, one clone showed significantly reduced sensitivity against in vivo glucocorticoid treatment which was accompanied by slow growth, identifying this clone as especially aggressive and challenging for treatment. Taken together, the present work established a novel approach to characterize challenging subclones regarding functional features and genetic characteristics which will help to develop efficient novel treatment approaches to eliminate aggressive cell clones in the future.Akute Leukämien bestehen aus heterogenen Zellpopulationen, die sich sowohl in genetischen als auch in funktionellen Eigenschaften unterscheiden können. Letztendlich ist die jeweils aggressivste Subpopulation eines Tumors entscheidend für die Prognose und den Krankheitsverlauf des Patienten. Ein besseres Verständnis von aggressiven Subklonen sowohl bezüglich Genotyp als auch funktionellem Phänotyp ist erforderlich, um neue Angriffspunkte für Chemotherapeutika zu finden und so die Prognose und Heilungsrate von Krebspatienten zu verbessern. Ziel der vorliegenden Arbeit war es, Einzelzellklone zu charakterisieren, um neue therapeutische Targets zu identifizieren. Dafür wurden primäre Tumorzellen von einem Mädchen mit akuter lymphatischer Leukämie (ALL) im ersten Rezidiv in immunsupprimierte Mäuse transplantiert und mit Lentiviren genetisch so modifiziert, dass sie ein rotes, ein grünes und ein blaues Fluoreszenzprotein in verschiedenen Mengen und Kombinationen exprimierten (RGB marking, (Weber et al., 2011)). Im Anschluss wurden Einzelzellklone der Leukämieprobe hergestellt, indem wenige RGB-gefärbte Xenograftzellen in Mäuse transplantiert wurden und dadurch individuell gefärbt Einzelzellen amplifiziert wurden. Die Identität der Zellen der Einzelzellklone wurde mittels LM-PCR bestätigt. Um aggressive Subklone aufzuspüren, wurden verschiedene Klone gemischt, zusammen in Mäuse transplantiert und in vivo Proliferationsassays und Chemoresistenzassays durchgeführt. Dabei konnten die Klone mittels Durchflusszytometrie anhand ihrer unterschiedlichen molekularen Farbmarkierungen klar voneinander unterschieden werden. Bei gemeinsamer Transplantation von Mischungen von verschiedenen Klonen zusammen in eine Maus wurden einige Klone von anderen überwachsen, was auf ein aggressives, langsames Wachstumsverhalten der überwachsenen Klone schließen lässt. Außerdem wurden zwei Klone gemeinsam in Mäuse transplantiert und diese Mäuse mit Glucocorticoiden behandelt. Dabei wies ein Klon eine erheblich geringere Sensitivität gegenüber in vivo Glucocorticoid-Behandlung in Kombination mit einem langsamen Wachstumsverhalten auf, was diesen Klon als besonders aggressiv und schwer zu behandeln identifizierte. Zusammenfassend wurde in der vorliegenden Arbeit eine neue Methode etabliert, um aggressive Subklone sowohl hinsichtlich funktioneller Besonderheiten als auch bezüglich genetischer Merkmale zu charakterisieren, was helfen wird, neue effiziente Behandlungsmethoden zu entwickeln, um aggressive Subklone in Zukunft besser eliminieren zu können

Zum Einfluss von sprachlichen Anweisungen auf Repräsentation und Leistung im Techniklernprozess

Meier C, Frank C, Gröben B. Zum Einfluss von sprachlichen Anweisungen auf Repräsentation und Leistung im Techniklernprozess. In: Amesberger G, Würth S, Finkenzeller T, eds. Zukunft der Sportpsychologie - zwischen Verstehen und Evidenz. Virtuelle Online-Tagung. 52. Jahrestagung der Arbeitsgemeinschaft für Sportpsychologie. Salzburg: Universität Salzburg; 2020: 104

An advanced preclinical mouse model for acute myeloid leukemia using patients' cells of various genetic subgroups and in vivo bioluminescence imaging.

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups

BLI is highly sensitive and reliable in single mice.

<p><b>(A)</b> 1x10⁵ t-PDX AML-372 cells were injected into two mice. At indicated days after cell injection, mice were monitored by BLI. Images of one representative mouse are shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s005" target="_blank">S5A Fig.</a> for further images. <b>(B)</b> BLI signals from the kinetic in <b>A</b> were quantified in both animals (diamonds); cells positive for both hCD45 and hCD33 in PB were analyzed in parallel (circles). <b>(C)</b> t-PDX AML-372 cells were injected into three mice per group at absolute numbers indicated; 1 and 8 days after cell injection, mice were monitored by BLI; images are shown of one representative mouse per group.</p

PDX AML cells allow genetic engineering without altering molecular sample characteristics.

<p><b>(A)</b> Scheme of the process of generating transgenic PDX (t-PDX) AML cells. PDX cells were transduced after first or second retransplantation cycle. <b>(B)</b> Scheme of the vector constructs. <b>(C)</b> Transduction rate in t-PDX AML cells after lentiviral transduction and cell amplification in mice was measured by FACS analysis of fluorochrome or NGFR expression. Each mark visualizes data obtained from a single transduction. Open mark: no transgenic cells were detectable. <b>(D)</b> Enrichment of transgenic cells using flow cytometry was measured using mCherry expression after cell amplification in mice. <b>(E)</b> Genetic engineering does not alter immunophenotype; primary cells, untransduced PDX cells after fourth retransplantation and enriched transgenic t-PDX cells were analyzed by multicolor flow cytometry; specific fluorescence intensity is depicted. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s003" target="_blank">S3C Fig.</a> for exemplary FACS plots of AML-372. Raw data is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s011" target="_blank">S3 Table</a>. <b>(F)</b> Genetic engineering does not markedly alter AML-specific mutations; genomic DNA was isolated out of primary cells, untransduced PDX cells and enriched transgenic t-PDX cells; VAF of mutations was profiled by targeted resequencing. <i>BCOR</i> (BCL-6 corepressor); <i>KRAS</i> (Kirsten rat sarcoma viral oncogene homolog); <i>NRAS</i> (neuroblastoma RAS viral oncogene homolog); <i>TP53</i> (tumor protein p53). Raw data is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s010" target="_blank">S2 Table</a>.</p

Engraftment and retransplantation of AML cells in NSG mice conserves genetic alterations of the primary sample.

<p>Primary AML patient samples and matched PDX cells, reisolated out of the BM (CD45 chimerism 80–99%) after first passage in NSG mice (PDX-0) or after 1 or 2 re-transplantation cycles (PDX-1/-2), were characterized by targeted resequencing of 43 AML-related genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s009" target="_blank">S1 Table</a>). Plots depict variant allele frequencies for each driver gene mutation found within the sample. a/b/c/d/f: PDX cells of three to five mice injected in parallel were analyzed. *: primary cells were frozen and thawed before injection. <i>BCOR</i> (BCL-6 corepressor); <i>CEBPA</i> (CCAAT/enhancer binding protein alpha); <i>DNMT3A</i> (DNA (cytosine-5)-methyltransferase 3 alpha); <i>FLT3</i> (Fms-related tyrosine kinase 3); ITD (internal tandem duplication); <i>KRAS</i> (Kirsten rat sarcoma viral oncogene homolog); <i>NPM1</i> (nucleophosmin-1); <i>NRAS</i> (neuroblastoma RAS viral oncogene homolog); <i>SRSF2</i> (serine/arginine-rich splicing factor 2); <i>TET2</i> (tet methylcytosine dioxygenase 2); <i>TP53</i> (tumor protein p53). Raw data is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s010" target="_blank">S2 Table</a>.</p

Engraftment of primary AML cells in NSG mice predicts reengraftment capacity.

<p>10⁷ fresh primary AML cells were injected and successfully engrafted in NSG mice; shown are characteristics of the first engraftment regarding passaging time (time period from cell injection until clinical signs of leukemia or latest between 20 and 25 weeks) <b>(A)</b>; percentage of cells positive for both hCD45 and hCD33 at time of sacrifice within mouse PB <b>(B)</b> and within BM (black cubes) or spleen (grey circles) <b>(C)</b>. Each mark visualizes data obtained from a single mouse. Open cubes indicate 0% human cells. Dotted line discriminates samples that reengrafted in secondary recipients from samples that did not. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s001" target="_blank">S1A Fig.</a> for exemplary FACS plots.</p