4 research outputs found
BLI is highly sensitive and reliable in single mice.
<p><b>(A)</b> 1x10<sup>5</sup> 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>10<sup>7</sup> 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