In vivo imaging of patient-derived ALL in mice. a

<p>Kinetics of leukemic growth; 5×10⁴ ALL-177 cells per mouse were injected into a group of 5 mice which were imaged repeatedly over time; a single representative mouse is shown; units in rainbow color scales are photons per second per cm2 per steradian (photons s⁻¹ cm²⁻¹ sr⁻¹); <b>b</b> Dose-response of leukemic growth; 1×10⁷–3×10⁴ serially diluted ALL-177 cells were injected into groups of 5 mice which were imaged 8 weeks after injection; a single representative mouse is shown for each group; <b>c, d</b>Good correlation of in vivo imaging to post mortem readout parameters; 12 mice were injected with 10⁵ ALL-50 cells/mouse; each week, 2 mice were imaged and sacrificed; organs were collected and cell suspensions prepared from half the organ and 1 femur and were analyzed by flow cytometry for expression of GFP/human CD38; the other half of the organ and the second femur were analyzed by immunohistochemistry for expression of terminal desoxynucleotidyl transferase (Tdt; arrows indicate leukemia cells); rare, +, ++,+++indicate a rough quantification of the number of leukemic cells per field; <b>c</b> shows 1 representative image per week and post mortem analysis of bone marrow; in all mice, mid-abdominal signals are unspecific. <b>d</b> correlates results from imaging and FACs analysis in each mouse; correlation coefficient 0,86; Supplemental Figure 6 shows data on spleens.</p

Generation of GLuc

<p><b>-expressing patient-derived ALL cells. a</b> Scheme of the lentiviral vector construct; arrow indicates start of transcription; RSV/5′LTR = hybrid of the Rous Sarcoma virus promoter and the U5 long terminal repeat from HIV-1 virus; EF1 P = constitutive elongation factor 1-alpha promoter; GLuc = membrane anchored form of the Gaussia luciferase (GLuc) enzyme fused to the transmembrane domain of CD8; T2A = “self-cleaving” 2A peptide from insect virus <i>Thosea asigna</i>; copGFP = green fluorescent protein cloned from copepod <i>Pontellina plumata</i>; 3′ΔLTR = HIV-1 virus long terminal repeat with a self-inactivating U3 deletion; <b>b, c</b>Transduction efficiency as determined by flow cytometry measurement of GFP expression after one round of amplification of transduced cells in mice; (<b>b</b>) in ALL-50 and ALL-199; (<b>c</b>) in all 9 patient-derived ALL samples studied; <b>d</b> Stability of biological characteristics of patient-derived ALL cells despite of lentiviral transduction; examples from data shown in detail in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052798#pone-0052798-g001" target="_blank">Figure 1</a>; comparison of ALL-199 cells before and after lentiviral transduction and sorting concerning drug-induced cell death after 48 hours in vitro (left panel), expression of cell surface markers (middle panel) and time to engraftment (right panel).</p

Quantification, quality parameters and visualization of minimal disease. a

<p>Procedure of quantification; images of the representative mouse shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052798#pone-0052798-g002" target="_blank">Figure 2a</a> were quantified over time; 3 different regions were analyzed which are indicated with red squares in the image: (i) sum of 2 regions at the lower extremities (legs); (ii) part of the abdomen (spleen); (iii) the whole mouse body (entire mouse); <b>b</b> Logarithmic relation between cell number injected and light emission; all mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052798#pone-0052798-g002" target="_blank">Figure 2b</a> were imaged after 10 weeks and images were quantified; shown is the mean +/− standard error of the mean (SEM); <b>c</b> Imaging reveals logarithmic growth over time and low assay variances; 5×10⁴ ALL-177 cells per mouse were injected into groups of 5 mice each in two independent experiments performed 2 months apart; mice were imaged repetitively over time and images quantified; shown is the mean of each group +/− SEM; <b>d</b> Imaging enables monitoring minimal disease; ALL-4S, ALL-177 (5×10⁴ cells/mouse) or ALL-199 (1×10⁴ cells/mouse) with expression level of GFP as indicated (middle panel, mean fluorescence intensity - MFI) were injected into 7 mice per group; engraftment was considered positive at light emission above 6×10⁴ photons per second in the ROI indicated (see Methods for details) (upper panel shows one representative mouse); 4 of 7 mice were sacrificed and analyzed; shown are the frequencies of leukemia cells in bone marrow as determined by quantitative real time polymerase chain reaction (qRT-PCR) as mean per group of mice; see also Supplemental Figure 10. In all mice, mid-abdominal signals are unspecific.</p

Imaging-based quantification of leukemia initiating cell frequencies. a

<p>Imaging visualizes dependence of leukemic growth on both time and cell numbers; experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052798#pone-0052798-g002" target="_blank">Figure 2b</a> was followed up over time in all groups injected with the different cell numbers; depicted is the quantification of imaging as mean of each group +/− SEM; <b>b</b> Imaging enables convenient determination of CSC frequencies; ALL-54 cells were freshly isolated from a mouse spleen, seeded at 10⁶ cells/ml and stimulated in vitro with PBS or TRAIL (1 µg/ml). After 48 hours, cells were serially diluted based on the cell concentration seeded at the beginning of the experiment and injected into groups of 2–3 mice. After 8 weeks, mice were imaged and analyzed for leukemic engraftment (defined using signals of legs only, identically as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052798#pone-0052798-g003" target="_blank">Figure 3D</a>; engraftment is indicated with a star); frequency of leukemia initiating cells was calculated out of engraftment rates using Poisson statistics. In all mice, mid-abdominal signals are unspecific.</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

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 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

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