Lipid features from extracted AML samples.

<p>(A) Saturation index of all lipids measured with shotgun MS. The analysis revealed significant changes of the mono- and polyunsaturated fatty acids in t(8;21) samples. (B) GP index of liposomes prepared from lipid extracts of various AML samples. GP is a measure of membrane order (higher GP equals more ordered membranes). t(8;21) samples exhibit lower GP values, indicating higher membrane fluidity.</p

Comparison of lipid profiles of various AML types.

<p>Each point on the volcano graph represents a single lipid specie. The analysis was performed using GraphPad PRISM 6.0.</p

Score plots of multivariate analysis by PCA.

<p>The t(8;21) samples are separated from the inv16 and AML-nk samples. Each patient sample was measured in triplicates and each point on the plot represents an individual measurement. The calculated sum of squares was 0.253 and 0.094 for the first and second component, respectively. The analysis was performed using SIMCA 14.0 software.</p

Description of patients.

<p>Description of patients.</p

Lipidomic approach for stratification of acute myeloid leukemia patients

<div><p>The pathogenesis and progression of many tumors, including hematologic malignancies is highly dependent on enhanced lipogenesis. De novo fatty-acid synthesis permits accelerated proliferation of tumor cells by providing membrane components but these may also alter physicochemical properties of lipid bilayers, which can impact signaling or even increase drug resistance in cancer cells. Cancer type-specific lipid profiles would permit us to monitor and interpret actual effects of lipid changes, potential fingerprints of individual tumors to be explored as diagnostic markers. We have used the shotgun MS approach to identify lipid patterns in different types of acute myeloid leukemia (AML) patients that either show no karyotype change or belong to t(8;21) or inv16 types. Differences in lipidomes of t(8;21) and inv(16) patients, as compared to AML patients without karyotype change, presented mostly as substantial modulation of ceramide/sphingolipid synthesis. Furthermore, between the t(8;21) and all other patients we observed significant changes in physicochemical membrane properties. These were related to a marked alteration in lipid saturation levels. The discovered differences in lipid profiles of various AML types improve our understanding of the pathobiochemical pathways involved and may serve in the development of diagnostic tools.</p></div

Sublethal irradiation and fresh donor cells reduces survival in NSG mice transplanted with AML-MNCs.

<p>(A) Kaplan-Meyer plot of mice that were transplanted with MNCs of AML patients. NSG mice were transplanted with freshly isolated MNCs with (fresh/irradiated; grey solid line) and without (fresh/non-conditioned; black solid line) previous conditioning, or thawed MNCs with (thawed/irradiated; grey dotted line) or without (thawed/non-conditioned; black dotted line) sublethal irradiation. The percentage of mice transplanted and available for bone marrow analysis is indicated below graph (BM analysis available). Using fresh donor cells and irradiation conditioning shortened the life span of the recipient mice (fresh/irradiated vs fresh/non-conditioned p = 0.007; thawed/irradiated vs thawed/non-conditioned p = 0.018; and fresh/irradiated vs thawed/irradiated p = 0.0054; fresh/non-conditioned vs thawed/non-conditioned p = 0.0051; Gehan-Breslow-Wicoxon Test). Rx = irradiation conditioning. (B) Plot shows the bone marrow chimerism (percentage of human CD45⁺ cells of total CD45⁺ cells) after the transplantation of AML samples from bone marrow (BM), peripheral blood (PB), and leukapheresis products (LPH) 12–16 weeks before. LPH-MNCs showed a significant increase in the bone marrow overall chimerism compared to BM-MNCs.</p

Characteristics of samples from 19 AML patients.

<p>BM = bone marrow, F = female, FAB = French-American-British-classification, FLT3-ITD = Internal tandem duplication of FLT3, FLT3-TKD = Tyrosine kinase domain mutation of FLT3, Int. = intermediate, LPH = leukapheresis, M = male, n.a. = not available, NPM1 = nucleophosmin1 mutated, PB = peripheral blood, Rx = irradiation.</p

Human CD3⁺ cells in NSG recipient mice are polyclonal activated T lymphocytes.

<p>(A) Human CD3⁺ cells are morphologically diverse. Sorted human CD45⁺CD3⁺ cells (sort purity: 98.6%) isolated from the bone marrow of a NSG recipient mouse that had received 5×10⁶–10⁷ freshly isolated MNCs from AML #10 three weeks before were cytospun and stained according to May-Grünwald Giemsa. (Graft phenotype in the recipient: bone marrow: 35% hCD45⁺; composition of human leukocytes: CD3⁺ = 93%, CD19⁺ = 0.8%, CD33⁺ = 0.8%; surface antigens on CD3⁺ cells: CD4⁺ = 80%, CD8⁺ = 14%, CD4⁺/CD8⁺ = 3%, TCRα/β⁺ = 99%, TCRγ/δ⁺ = 0.02%). Photograph is representative for 6 different AML samples. Scale bar indicates a section of 10 micrometers. (B) Human CD3⁺ cells are identified as T lymphocytes by the surface expression of the T cell receptors (TCRs) and co-receptors. Graph shows a summary of the expression of TCRα/β or TCRγ/δ, and the expression of CD4 and CD8 on CD3⁺ cells the bone marrow of NSG mice that were transplanted with AML-MNCs as described in (A) (Within CD3⁺ cells: TCRγ/δ: 5.0±15.2%, TCRα/β: 89.2±22.1%; Within TCRα/β⁺ cells: CD4⁺ CD8⁻: 66.2±23.6%, CD4⁻ CD8⁺: 20.2±19.2%, CD4⁺ CD8⁺: 9.3±14.5%.) (C) Plot shows the frequency of indicated Vβ segments used in human α/β T cell receptors on T cells in the blood of healthy controls (‘in man’) and in the bone marrow of NSG mice that had received MNCs from AML patients as described in (A, ‘in mice’). Frequencies based on human CD45⁺ CD3⁺ cells are shown. (D) Bar graph shows the frequency of human T lymphocytes (hCD45⁺CD3⁺) that express CD25 and CD69 in blood, bone marrow and spleen of NSG mice that were transplanted with 10⁷ MNCs from AML patients 9.7±2.2 weeks before. N = 53 for all groups. (E) Bar graph shows the frequency of T lymphocytes that express CD25 and CD69 in the peripheral blood (4.7±0.3% of T lymphocytes) and bone marrow (21.9±10.2%) of healthy donors. N = 3 for all groups. (F) Maximum blood chimerism was determined after the transplantation of titrated numbers of CD2⁺ T cells that were sorted from the peripheral blood of a healthy donor. Donor-cell chimerism ≥0.1% hCD45⁺ (considered T cell engraftment, left y-axis) was reached only if ≥10⁵ CD2⁺ cells were transplanted. Virtually all engrafted human leukocytes expressed CD3 (% hCD45⁺CD3⁺, right y-axis). Mean and standard deviation are shown. (G) Plot depicts donor-cell chimerism in the bone marrow of NSG recipient mice as described in (D). Mean and standard deviation are shown.</p

Depletion of CD3⁺ and CD19⁺ cells from the graft prevents xGvHD symptoms, and augments AML engraftment.

<p>Unconditioned NSG mice were transplanted with equal numbers (0.15–0.6×10⁷ cells) of thawed MNCs from AML #7–9 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060680#pone-0060680-t001" target="_blank">Table 1</a>) with or without previous depletion of CD3⁺ and CD19⁺ lymphocytes (CD3⁻CD19⁻MNC and MNC, respectively). Mice were sacrificed for bone marrow analysis 12 weeks after transplantation, or when detoriation of health occurred, and analyzed by flow cytometry. AML samples #7–9 showed a common, characteristic phenotype of <i>Npm1</i>-mutated AML with positivity for CD33 and CD117, but lack of CD34 expression. (A) Dot plots show expression of CD3 versus CD33 (left column), CD34 versus CD117 (middle), and side scatter (SSC) versus CD45 (right column) on human CD45⁺ MNCs from a healthy donor (top), from AML patient #7 (second row from top), and from the bone marrow of NSG recipient mice that were transplanted with either MNCs (second row from bottom) or CD3⁻CD19⁻MNCs (bottom) from AML #7 twelve weeks before. Data representative for all AML samples #7–9 analyzed. (B-E) Plots show the mean donor-cell chimerism (B, % hCD45⁺; MNC: 33.3±27%, CD3⁻CD19⁻MNC: 22.4±30.9%), the frequency of CD19⁺ cells (C, MNC: 18.5±26.0%, CD3⁻CD19⁻MNCs: 12.3±14.3%), or CD3⁺ cells (D, MNC: 42.4±43.5%, CD3⁻CD19⁻MNCs: 0.02±0.02%, p = 0.0247), or CD33⁺ cells (E, MNC: 35.9±36.3%, CD3⁻CD19⁻MNCs: 85.0±17.8%, p = 0.0087) within donor-derived leukocytes (hCD45⁺) in the bone marrow of recipient mice 6–12 weeks after transplantation with AML-samples #7–9 (MNC: 33.3±27%, CD3⁻CD19⁻MNC: 22.4±30.9%).</p

Intra- and inter-sample heterogeneity in the engraftment of MNCs from AML patients.

<p>(A) Dot plots show the frequency of human leukocytes in the bone marrow of three NSG recipient mice that were transplanted with MNCs from the blood from AML patient #9 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060680#pone-0060680-t001" target="_blank">Table 1</a>), 7–10 weeks before (left column). Human cells were further analyzed for the cell surface expression of CD3 versus CD33, and CD33 versus CD19 (middle and right column, respectively). (B) Dot plots show human leukocytes in the bone marrow of NSG recipient mice that were transplanted with MNCs from LPH, BM or PB from AML patients #18, #14, and #12 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060680#pone-0060680-t001" target="_blank">Table 1</a>), respectively. Human leukocytes were analyzed for the expression of CD3, CD19 and CD33 six weeks (AML #18 and #14) or ten weeks (AML #12) after transplantation. (C) Frequency of CD3, CD19 or CD33 positive human leukocytes in the blood (PB, asterix), bone marrow (BM, circles) and spleen (diamonds) of NSG mice are presented as percentage of human CD45⁺ cells. The number of AML-samples (AML), independent experiments (exp.), and recipient mice (mice) is indicated.</p