Journal: Blood Cancer Journal

Article Title: Induction of NK cell reactivity against acute myeloid leukemia by Fc-optimized CD276 (B7-H3) antibody

doi: 10.1038/s41408-024-01050-6

Figure Lengend Snippet: A CD276 mRNA expression in AML bone marrow (BM) ( n = 350) and AML peripheral blood (PB) ( n = 318) and healthy BM ( n = 16) samples were analyzed using the public data sets of the BeatAML program . B Flow cytometric analysis of surface CD276 expression on the indicated AML cell lines using chimeric mouse anti-human 8H8 mAb against CD276 and corresponding isotype control. Example histograms of one representative experiment out of three with similar results are shown. C The gating strategy for two representative patient-derived AML samples is outlined as follows: singlets, viable (7-AAD - ), leukemic blast marker (CD33 + ), and the percentage of CD276 expression. D CD276 expression on AML patient blasts ( n = 68) is shown as percentage of CD276 + AML blasts and E as SFI levels. Expression levels above SFI of 1.5 and 10% for positive cells were considered as positive expression (indicated by the dotted line and shown by the corresponding pie chart). F Schematic representation of the generated CD276 antibody with a modified Fc part optimized for increased affinity to CD16 (8H8_SDIE) and the corresponding control mAb (MOPC_SDIE). G Titration of the 8H8_SDIE mAb indicated AML cell lines and H on AML patient sample analyzed by flow cytometry. MOPC_SDIE was used as an isotype control. The example data show the MFI values of one representative experiment out of a total of three with comparable results.

Article Snippet: The AML cell lines (EOL-1, HL-60, Kasumi-1, KG-1a, MOLM-13, MV4-11, NB-4, Nomo-1, SKM-1, THP-1, U937) were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Control, Derivative Assay, Marker, Generated, Modification, Titration, Flow Cytometry

Journal: Blood Cancer Journal

Article Title: Induction of NK cell reactivity against acute myeloid leukemia by Fc-optimized CD276 (B7-H3) antibody

doi: 10.1038/s41408-024-01050-6

Figure Lengend Snippet: PBMC from healthy donors were co-cultured with the indicated leukemic cells in the presence or absence of 8H8_SDIE mAb or MOPC_SDIE control (both 1 µg/mL). A Targeted lysis of AML cells was determined by Europium-based cytotoxicity assays after 2 h of incubation. The left panel shows example data with THP-1 cells and a PBMC donor at different E:T ratios. The right panel shows pooled data from EOL-1, U937 and THP-1 cell lines with different PBMC donors ( n = 6-9) (E:T 80:1). B Lysis of AML cell lines with different PBMC donors ( n = 3-4) was analyzed by flow cytometry after 24 h (E:T 20:1). C Lysis of the different AML cell lines with PBMC from healthy donors ( n = 3-4) was analyzed by flow cytometry after 72 h (E:T 20:1). D Survival of AML cell lines was determined using a live cell imaging system. THP-1 cells were cultured with PBMC from healthy donors ( n = 4) (E:T 80:1) for 96 h. Dead target cell areas were normalized to the initial target cell area’s initial measurement at T = 0 h.

Article Snippet: The AML cell lines (EOL-1, HL-60, Kasumi-1, KG-1a, MOLM-13, MV4-11, NB-4, Nomo-1, SKM-1, THP-1, U937) were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and American Type Culture Collection (Manassas, VA, USA).

Techniques: Cell Culture, Control, Lysis, Incubation, Flow Cytometry, Live Cell Imaging

Journal: Blood Cancer Journal

Article Title: Induction of NK cell reactivity against acute myeloid leukemia by Fc-optimized CD276 (B7-H3) antibody

doi: 10.1038/s41408-024-01050-6

Figure Lengend Snippet: A Immunocompromised NSG mice ( n = 4/group) were injected with human PBMC (20 × 10 6 cells, i.v.) and treated with an CD3 antibody (clone UCHT1, 20 µg, i.v.) as control or 8H8_SDIE (20 µg, i.v.). Blood was collected 24 h after injection and serum levels of IFNγ, IL-2, TNF, IL-6 were measured by Legendplex. B Luciferase-expressing U937 cells (1 × 10 6 ) were injected intravenously into NSG mice ( n = 5/group) followed by PBMC injection (40 × 10 6 , i.v.) and 8H8_SDIE (20 µg, i.v.) on the next day. AML tumor burden was monitored over time by bioluminescence imaging. The left panel shows exemplary bioluminescence images of mice from the indicated treatment groups on days 7 and 14. The middle panel shows the quantitative analysis of AML tumor burden measured by luminescence for all treatment groups on days 7 and 14. The right panel shows the AML tumor burden measured twice a week over the 2-week period.

Article Snippet: The AML cell lines (EOL-1, HL-60, Kasumi-1, KG-1a, MOLM-13, MV4-11, NB-4, Nomo-1, SKM-1, THP-1, U937) were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and American Type Culture Collection (Manassas, VA, USA).

Techniques: Injection, Control, Luciferase, Expressing, Imaging

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: Cell Death & Disease

Article Title: LncRNA BC200/miR-150-5p/MYB positive feedback loop promotes the malignant proliferation of myelodysplastic syndrome

doi: 10.1038/s41419-022-04578-2

Figure Lengend Snippet: A In SKM-1 and MDS-L cells, the transcriptional level of BC200 was downregulated by two shRNAs. B–D MDS cells proliferation was detected by CCK-8, EdU incorporation (Scale bar, 100 μm) and colony formation assays. It revealed that BC200 knockdown significantly suppressed SKM-1 and MDS-L cell proliferation. E , F Knockdown of BC200 led to G0/G1 arrest in both SKM-1 and MDS-L cells. G Apoptosis assay showed that the percentage of apoptotic MDS cells was not affected by BC200 knockdown. H–J Human primary BMMCs were treated with BC200 siRNAs for 72 h, and cell proliferation was analyzed by the CCK-8 assay. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: Human MDS cell lines (SKM-1 and MDS-L) and HEK293T cell lines were purchased from ATCC (Manassas, VA, USA).

Techniques: CCK-8 Assay, Knockdown, Apoptosis Assay

Journal: Cell Death & Disease

Article Title: LncRNA BC200/miR-150-5p/MYB positive feedback loop promotes the malignant proliferation of myelodysplastic syndrome

doi: 10.1038/s41419-022-04578-2

Figure Lengend Snippet: A RNA FISH assays for BC200 in SKM-1 cells. Stain the nucleus with DAPI. Scale bar, 25 μm. B BC200, U6, and ACTB expression in the RNA extracted from the cytoplasm and nucleus of MDS cells by qRT-PCR. C The ceRNA regulatory network of BC200 and three miRNAs containing the binding site of BC200. D Transcriptional level of miR-150-5p was detected in sh-BC200 MDS cells. E Upper: complementary sequence between miR-150-5p and BC200-wt. The putative binding sites of miR-150-5p in BC200-mt. Lower: Luciferase activity was measured in HEK293T cells cotransfected with miR-150-5p mimics and wt or mt BC200 vector. F RIP assays of the enrichment of Ago2 on BC200 and miR-150-5p relative to IgG in MDS-L cells. The relative expression levels of BC200 and miR-150-5p were detected by qRT-PCR. G Detection of BC200 or miR-150-5p by using qRT-PCR in the sample pulled down by biotinylated BC200 and Random probe. Input was used for normalization. H The correlation among BC200, miR-150-5p, and Ago2 was determined by using Ago2 antibody to detect cell lysates utilizing the sample pulled down by biotinylated BC200 and Random probe. I CCK-8 assay rescue experiment showed that cell proliferation reduced by si-BC200 could be increased by miR-150-5p inhibitor in MDS cells. J CCK-8 assay rescue experiment showed that cell proliferation stimulated by overexpression of BC200 could be suppressed by miR-150-5p mimics in MDS cells. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: Human MDS cell lines (SKM-1 and MDS-L) and HEK293T cell lines were purchased from ATCC (Manassas, VA, USA).

Techniques: Staining, Expressing, Quantitative RT-PCR, Binding Assay, Sequencing, Luciferase, Activity Assay, Plasmid Preparation, CCK-8 Assay, Over Expression

Journal: Cell Death & Disease

Article Title: LncRNA BC200/miR-150-5p/MYB positive feedback loop promotes the malignant proliferation of myelodysplastic syndrome

doi: 10.1038/s41419-022-04578-2

Figure Lengend Snippet: A MYB was the top candidate target containing the complementary site for the seed region of miR-150-5p in the TargetScan database. B , C SKM-1 and MDS-L cells were transfected with miR-150-5p mimics or inhibitors for 48 h, and MYB mRNA levels were detected by qRT-PCR. D , E MYB protein expression in both SKM-1 and MDS-L cells transfected with miR-150-5p mimics or inhibitor for 48 h was detected by western blotting. F Upper: putative MYB binding sites on the promoter region of miR-150-5p and their corresponding mutant binding sites. Lower: luciferase activity in HEK293T cells cotransfected with miR-150-5p or control mimics and luciferase reporters containing the wt or mt MYB 3'-UTR. G CCK-8 assay detected the effect of MYB overexpression and miR-150-5p mimics on SKM-1 and MDS-L cell proliferation activity after 72 h. H CCK-8 assay detected the effect of MYB knockdown and miR-150-5p inhibitor on SKM-1 and MDS-L cell proliferation activity after 72 h. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: Human MDS cell lines (SKM-1 and MDS-L) and HEK293T cell lines were purchased from ATCC (Manassas, VA, USA).

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Control, CCK-8 Assay, Over Expression, Knockdown

Journal: Cell Death & Disease

Article Title: LncRNA BC200/miR-150-5p/MYB positive feedback loop promotes the malignant proliferation of myelodysplastic syndrome

doi: 10.1038/s41419-022-04578-2

Figure Lengend Snippet: A The mRNA levels of MYB were measured in sh-BC200 MDS cells. B The protein expression levels of MYB were measured in MDS cells transfected with two different BC200 shRNAs. C , D qRT-PCR and western blotting were used to measure the MYB mRNA and protein levels in SKM-1 and MDS-L cells transfected with BC200 plasmid (ov-BC200) for 48 h. E , F Western blot analysis of MYB in MDS cells cotransfected with miR-150-5p mimics and BC200 siRNA or miR-150-5p inhibitors and BC200 plasmid for 48 h. G CCK-8 assay indicated the effect of BC200 overexpression and MYB suppression on SKM-1 and MDS-L cell proliferation activity after 72 h. H CCK-8 assay indicated the effect of BC200 knockdown and MYB upregulation on SKM-1 and MDS-L cell proliferation activity after 72 h. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: Human MDS cell lines (SKM-1 and MDS-L) and HEK293T cell lines were purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Plasmid Preparation, CCK-8 Assay, Over Expression, Activity Assay, Knockdown

Journal: Cell Death & Disease

Article Title: LncRNA BC200/miR-150-5p/MYB positive feedback loop promotes the malignant proliferation of myelodysplastic syndrome

doi: 10.1038/s41419-022-04578-2

Figure Lengend Snippet: A HEK293T cells were cotransfected with GV238-BC200 and MYB siRNAs for 48 h. B The MYB binding sitein BC200 predicted by JASPAR matrix models. C Upper: putative MYB binding sites BC200 promoter region and design of the indicated primers. Lower: ChIP assays of the enrichment of MYB on the BC200 promoter relative to control IgG in MDS-L cells. D The expression level of BC200 was detected in MDS cells transfected with two different MYB siRNAs. E , F SKM-1 and MDS-L cells were treated with the indicated siRNAs or plasmids for 72 h. The cell proliferative was measured by CCK-8 assay. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: Human MDS cell lines (SKM-1 and MDS-L) and HEK293T cell lines were purchased from ATCC (Manassas, VA, USA).

Techniques: Binding Assay, Control, Expressing, Transfection, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: LncRNA BC200/miR-150-5p/MYB positive feedback loop promotes the malignant proliferation of myelodysplastic syndrome

doi: 10.1038/s41419-022-04578-2

Figure Lengend Snippet: A SKM-1 cells treated with different BC200 shRNAs ( n = 4) or control shRNA ( n = 4) were inoculated subcutaneously into NCG mice. Tumor growth curves showed that sh-BC200 group led to tumor growth restriction in mice. Scale bar: 1 cm. B Tumor growth curves showed that sh-BC200 group suppressed tumor growth compared with sh-control group. C The subcutaneous tumors were harvested and weighed on the 26th day after implantation. Data are shown as mean ± SEM. D The NCG mice grew tumors on the fourteenth day after implantation and began to be weighed. E Ki-67 immunostaining of xenograft tissues collected from the BC200-knockdown group and control group. Scale bar: 50 μm. F–H qRT-PCR was performed on xenograft tissues and was subjected to measure BC200, MYB, and miR-150-5p expression. I Plot of CD123 vs CD34 expression of BM cells of MDS or normal mice being analyzed. J White blood cells (WBCs), red blood cells (RBCs), hemoglobin (HGB), hematocrit (HCT), and platelets (PLTs) counts of MDS or normal mice. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human MDS cell lines (SKM-1 and MDS-L) and HEK293T cell lines were purchased from ATCC (Manassas, VA, USA).

Techniques: Control, shRNA, Immunostaining, Knockdown, Quantitative RT-PCR, Expressing