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