Journal: bioRxiv

Article Title: Laminin Receptor Characterization in Acute Myeloid Leukemia: Integrin α7β1 Defines non-Leukemic Stem Cells with Migratory Potential

doi: 10.1101/2024.03.29.587290

Figure Lengend Snippet: (A) Comparison of gene expression between healthy BM samples (GTEx, n=70) and AML samples (TCGA, n=173) using Gepia . ITGA6 and ITGA7 are overexpressed in AML. (B) Flow cytometry analysis of laminin receptor expression on healthy mobilized CD34+ CD38-HSPC (n=5), primary CD33+ AML cells (n=60) and AML cell lines (n=7) shows an overexpression of integrin α6 and α7 in AML, depicted as mean +/-SD. (C) Exemplary flow cytometry staining of two selected patients showing laminin receptor surface expression on primary AML cells.

Article Snippet: AML cell lines (Kasumi-1, MOLM-13, NOMO-1, SKM-1, THP-1) were obtained from DSMZ and human umbilical cord endothelial cells (HUVEC) from ATCC.

Techniques: Comparison, Gene Expression, Flow Cytometry, Expressing, Over Expression, Staining

Journal: bioRxiv

Article Title: Laminin Receptor Characterization in Acute Myeloid Leukemia: Integrin α7β1 Defines non-Leukemic Stem Cells with Migratory Potential

doi: 10.1101/2024.03.29.587290

Figure Lengend Snippet: (A) Flow cytometric analysis shows heterogeneous laminin receptor surface expression between AML cell lines. HNT-34, SKM-1 and THP-1 display the expression of the most laminin receptors as opposed to MOLM-13, which is negative for all laminin receptors analyzed. (B) Western blotting of laminin receptors confirms the expression patterns of integrin α3, α6 and BCAM as seen by flow cytometry. Expression of integrin α7 is very pronounced for Kasumi-1 in Western blotting, but not measurable via flow cytometry. (C) ImageStream analysis of intracellular and extracellular integrin α7 shows expression on the cell surface in SKM-1, but only intracellular expression in Kasumi-1. (D) Comparison between laminin receptor expression on BM and PB primary AML cells reveals higher integrin α7 expression on PB cells. (E) Comparison between laminin receptor expression on AML samples positive for CD34 (> 5 % CD34+ cells) and negative for CD34 (< 1 % CD34+ cells) shows higher surface expression of integrin α7 on CD34-AML samples, whereas integrin α6 and BCAM are higher expressed in CD34+ AML.

Article Snippet: AML cell lines (Kasumi-1, MOLM-13, NOMO-1, SKM-1, THP-1) were obtained from DSMZ and human umbilical cord endothelial cells (HUVEC) from ATCC.

Techniques: Expressing, Western Blot, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Laminin Receptor Characterization in Acute Myeloid Leukemia: Integrin α7β1 Defines non-Leukemic Stem Cells with Migratory Potential

doi: 10.1101/2024.03.29.587290

Figure Lengend Snippet: (A) Representative images showing adhesion assays with primary AML cells and the AML cell line THP-1 on different laminin isoform coatings. Plastic dishes were coated with single laminin isoform droplets and unspecific cell adhesion to plastic was blocked with BSA. Laminin specific adhesion is seen in the circular droplet region. Brightfield images were acquired with a microscope (Zeiss, Primovert). (B) Schematic drawing of laminin-511 and its binding specificities. All four laminin receptors have been described to bind to the C-terminal end of laminin-511 ( , ) (C) Phenotyping of adherent and non-adherent cell fractions, marker expression on adherent cells was normalized to the expression on non-adherent cells within each sample. Adherent cells show a higher expression of integrin α3 and α6 and a reduced expression of NKG2DL. (D) Primary AML cells adhere to laminin-511 but not to laminin-211 or laminin-111. (E) Proliferation assays with the SKM-1 AML cell line on different laminin coatings or without coating (control). Laminin-211 coating slightly increases cell proliferation (n=3). (F) Blocking antibodies against integrin α6 and integrin β1 reduce adhesion of primary AML cells to laminin-511 whereas blocking antibodies against integrin α7 and BCAM have no effect on cell adhesion.

Article Snippet: AML cell lines (Kasumi-1, MOLM-13, NOMO-1, SKM-1, THP-1) were obtained from DSMZ and human umbilical cord endothelial cells (HUVEC) from ATCC.

Techniques: Microscopy, Binding Assay, Marker, Expressing, Control, Blocking Assay

Journal: bioRxiv

Article Title: Laminin Receptor Characterization in Acute Myeloid Leukemia: Integrin α7β1 Defines non-Leukemic Stem Cells with Migratory Potential

doi: 10.1101/2024.03.29.587290

Figure Lengend Snippet: Representative images of adhesion assays to different laminin isoform coatings. (A) Adhesion assays of AML cell lines show strong adhesion to laminin-511 and weak adhesion to laminin-332. All tested cell lines but MOLM-13 adhere to laminin-511. (B) Adhesion assays of primary AML cells show adhesion to laminin-511 in some samples, while other patient samples do not adhere. (C) Gene knockouts of integrin α6 and BCAM were generated in the AML cell lines Kasumi-1 and SKM-1 and the absence of surface expression of the respective marker was validated using flow cytometry. Deletion of integrin α6 reduces adhesion to laminin-511, but deletion of BCAM does not affect adhesion. (D) Flow cytometry data of primary AML samples analyzed for integrin α6 and α7 co-expression in non-LSC (NKG2DL+) and LSC populations (NKG2DL-). (E) QRT-PCR analysis of ITGA7 isoforms in primary AML cells (n=11) and AML cell lines (n=7). The isoform α7X2 is higher expressed than α7X1.

Article Snippet: AML cell lines (Kasumi-1, MOLM-13, NOMO-1, SKM-1, THP-1) were obtained from DSMZ and human umbilical cord endothelial cells (HUVEC) from ATCC.

Techniques: Generated, Expressing, Marker, Flow Cytometry, Quantitative RT-PCR

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, Single Cell