Journal: FEBS Open Bio

Article Title: Modulators of histone demethylase JMJD1C selectively target leukemic stem cells

doi: 10.1002/2211-5463.13054

Figure Lengend Snippet: Identification of JDM‐7 (#7) as LSCs specific repressor. The effect of JDM‐7 on colony formation of MLL r AL cell lines MOLM‐13, MV4‐11, THP‐1 and cord blood cells. One thousand MOLM‐13, 2000 MV4‐11 cells and 2000 THP‐1 cells were cultured in methylcellulose medium (H4100; Stemcell Technologies) plus 10% fetal bovine serum with or without JDM‐7 for the days and compound concentrations indicated. Twenty‐five thousand cord blood cells were cultured in methylcellulose medium (H4434; Stemcell Technologies) with or without JDM‐7 for the days and compound concentrations indicated. Colonies were counted. Three ( n = 3) replicated experiments were performed, a two‐sided Student’s t ‐test was used for statistical analysis and P < 0.05 was considered statistically significant. Error bars indicate the SD. * P < 0.05. BFU, burst‐forming unit‐erythroid; CFU‐E, colony‐forming unit‐erythroid; CFU‐GM, colony‐forming unit‐granulocyte, macrophage.

Article Snippet: Human MOLM‐13, MV4‐11 and THP‐1 cells were plated in methylcellulose media (Methocult H4100; Stemcell Technologies, Cologne, Germany) supplemented with 10% fetal bovine serum in triplicates at a cell dose of 1000 per plate for MOLM‐13 and 2000 for MV4‐11 and THP‐1.

Techniques: Cell Culture

Journal: FEBS Open Bio

Article Title: Modulators of histone demethylase JMJD1C selectively target leukemic stem cells

doi: 10.1002/2211-5463.13054

Figure Lengend Snippet: Tadalafil represses the CFU of leukemia cells. The effect of tadalafil on colony formation of MLL r AL cell lines MOLM‐13, MV4‐11, THP‐1 and cord blood cells. One thousand MOLM‐13, 2000 MV4‐11 cells and 2000 THP‐1 cells were cultured in methylcellulose medium (H4100; Stemcell Technologies) plus 10% fetal bovine serum with or without tadalafil for the days and compound concentrations indicated. Twenty‐five thousand cord blood cells were cultured in methylcellulose medium (H4434; Stemcell Technologies) with or without tadalafil for the days and compound concentrations indicated. Colonies were counted. Three ( n = 3) replicated experiments were performed, a two‐sided Student's t ‐test was used for statistical analysis and P < 0.05 was considered statistically significant. Error bars indicate the SD. * P < 0.05. T10, T25, T50 and T100 indicates tadalafil at 10, 25, 50 and 100 μ m . BFU, burst‐forming unit‐erythroid; CFU‐E, colony‐forming unit‐erythroid; CFU‐GM, colony‐forming unit‐granulocyte, macrophage.

Article Snippet: Human MOLM‐13, MV4‐11 and THP‐1 cells were plated in methylcellulose media (Methocult H4100; Stemcell Technologies, Cologne, Germany) supplemented with 10% fetal bovine serum in triplicates at a cell dose of 1000 per plate for MOLM‐13 and 2000 for MV4‐11 and THP‐1.

Techniques: Cell Culture

Journal: Cancer Immunology Research

Article Title: Broad Cytotoxic Targeting of Acute Myeloid Leukemia by Polyclonal Delta One T Cells

doi: 10.1158/2326-6066.cir-18-0647

Figure Lengend Snippet: Figure 2. Clonal DOT-cell reactivity against AML cells. A and B, In vitro killing of AML KG-1 cells by DOT-cell clones generated from single Vd1þ T cells sorted from healthy donors. Cells were coincubated for 3 hours at 10:1 (E:T) ratio and then analyzed by Annexin V staining (percentage of positive events among prelabeled KG-1 cells). Each bar represents killing of KG-1 cells upon coincubation with individual clones. Dashed red line represents the mean basal tumor cell death (without DOT cells). In B, either anti-Vd1þ TCR-specific mAb or isotype control was added to the cultures. Shown are the clones where the blockade led to clearer reduction in KG-1 targeting. Data represent the average of two technical replicates and are derived from 4 independent healthy donors (HD). C, Real-time PCR assessing B7-H6 mRNA in parental (B7-H6þ/þ) and CRISPR/Cas9-manipulated (B7-H6/) AML HEL cell lines. D, In vitro killing of B7-H6þ/þ or B7-H6/ AML HEL cells by bulk DOT cells produced from 3 healthy donors (tested in technical duplicates). Cells were coincubated for 3 hours at 10:1 (E:T) ratio and then analyzed by Annexin V staining (shown are percentages of positive events among prelabeled HEL cells). Indicated are mean þ SEM (, P < 0.001).

Article Snippet: AML cell targeting in vitro and in vivo AML cell lines (THP-1, HEL, AML-193, MV4-11, HL-60, U-937, OCI-AML3, Kasumi-1, and KG-1) were obtained from and authenticated by the German Resource Center for Biologic Mate- rial (DSMZ); and used at passages p3–p8.

Techniques: In Vitro, Clone Assay, Generated, Staining, Control, Derivative Assay, Real-time Polymerase Chain Reaction, CRISPR, Produced

Journal: Cancer Immunology Research

Article Title: Broad Cytotoxic Targeting of Acute Myeloid Leukemia by Polyclonal Delta One T Cells

doi: 10.1158/2326-6066.cir-18-0647

Figure Lengend Snippet: Figure 3. DOT cells target multiple AML cell types but not healthy leukocytes. In vitro killing assays with DOT cells produced from 3–4 healthy donors, coincubated for 3 hours at 10:1 (E:T) ratio with the indicated AML cell lines (A), primary AML samples (B), or normal leukocyte populations FACS-sorted from the peripheral blood (C). In A, the dashed red line represents the mean basal tumor cell death; and in B, CTR refers also to tumor cells alone (without DOT cells). Experiments were performed with technical triplicates. In vivo AML targeting by DOT cells. DOT cells (3 injections of 2 107 cells, see Supplementary Fig. S5A–S5C) were transferred to NSG mice (n ¼ 6 CTR, 7 DOT-treated mice) preinjected with KG-1 AML cells (D–E); or NSGS mice (n ¼ 5 CTR, 5 DOT-treated mice) bearing primary AML cells (F–G; patient-derived xenograft, PDX). Tumor burden was assessed in the blood and liver one week after the last DOT-cell transfer (D); or through weekly bleedings (F). Indicated are mean þ SEM, , P < 0.05; , P < 0.001; , P < 0.0001. Animals were sacrificed when advanced disease symptoms (such as back leg paralysis) were observed. Survival curves are presented in panels E (P < 0.05) and G (P < 0.01).

Article Snippet: AML cell targeting in vitro and in vivo AML cell lines (THP-1, HEL, AML-193, MV4-11, HL-60, U-937, OCI-AML3, Kasumi-1, and KG-1) were obtained from and authenticated by the German Resource Center for Biologic Mate- rial (DSMZ); and used at passages p3–p8.

Techniques: In Vitro, Produced, In Vivo, Derivative Assay

Journal: Cancer Immunology Research

Article Title: Broad Cytotoxic Targeting of Acute Myeloid Leukemia by Polyclonal Delta One T Cells

doi: 10.1158/2326-6066.cir-18-0647

Figure Lengend Snippet: Figure 4. DOT cells (re-)target chemotherapy-resistant AML. Comparison of the in vitro anti-AML activity of DOT cells and standard chemotherapy. A, DOT cells and standard AML chemotherapy (doxorubicin plus cytarabine) protocols were tested against chemotherapy-na€ve (wild type, wt) or chemo-relapsed (CR, regrown after >99% HEL cell elimination) AML cells. Shown are the percentages of Annexin Vþ HEL cells after 3 hours of treatment. B, Number of AML HEL cells before and after 72 hours of treatment with DOT cells (at 5:1 E:T ratio). Surviving cells (<1%) were resorted and allowed to regrow, thus generating the DOT-treated (DT) samples of (C–E). C, DOT cells were coincubated for 3 hours with nontreated (NT) or previously DOT-treated (DT) AML HEL cells at 5:1 or 10:1 (E:T) ratios. Shown are the percentages of Annexin Vþ HEL cells. D, Number of barcoded AML single-cell lineages in non-treated (NT), chemotherapy-treated (CT), or DOT-treated (DT) AML HEL cells. E, Pearson correlation for distribution of barcoded AML single-cell lineages between different treatments. Red, yellow, and green dashed lines represent low, medium, and high correlations, respectively. Indicated are mean þ SEM (, P < 0.01; , P < 0.001; , P < 0.0001).

Article Snippet: AML cell targeting in vitro and in vivo AML cell lines (THP-1, HEL, AML-193, MV4-11, HL-60, U-937, OCI-AML3, Kasumi-1, and KG-1) were obtained from and authenticated by the German Resource Center for Biologic Mate- rial (DSMZ); and used at passages p3–p8.

Techniques: Comparison, In Vitro, Activity Assay