Journal: Cancers

Article Title: FUT9-Driven Programming of Colon Cancer Cells towards a Stem Cell-Like State

doi: 10.3390/cancers12092580

Figure Lengend Snippet: Identification of gene regulatory networks linking FUT9 expression to stemness in glyco-engineered colon cancer cells. ( A ) RT-PCR-based assessment of mRNA levels of murine fucosyltransferases (FUT) upon transcriptional activation of the Fut9 gene in MC38 cells using the CRISPR-dCas9-VPR technology. Expression was normalized to the housekeeping gene Gapdh ( M. musculus ). Differences (fold change) relative to MC38-MOCK cells are depicted in triplicates. Data representative of two independent experiments. ( B ) Western blot analysis for FUT9 in MC38-glycovariants. Data representative of two independent experiments. ( C ) Flow cytometric analysis of the Lewis x expression on the surface of MC38 glyco-engineered cells. Histograms representative of two independent experiments. ( D ) Representative images of anti-Lewis x immunofluorescence staining in MC38-MOCK and MC38-FUT9 cells. DAPI staining represents nucleic acid (nuclear) staining. Scale bar 10 μm. ( E ) Venn diagram depicting the number of differentially expressed genes (DEGs) between MC38-WT vs. MC38-MOCK and MC38-FUT9 vs. MC38-MOCK cells identified by RNA-seq analysis. In MC38-FUT9 cells compared to MC38-MOCK cells, 1709 genes were upregulated and 1874 genes were downregulated. ( F ) The top 3 of the predicted transcription factors (TFs) identified by the RNA-seq-based regulon analysis (iRegulon) corresponding to the upregulated genes (top panel) and the downregulated genes (lower panel) in MC38-FUT9 cells. NES represents the normalized enrichment score. The number of the predicted direct targets among DEGs and the significantly enriched motifs corresponding to each regulator is provided in the table. More details on the motifs and TF-associated genes can be found in . ( G ) Normalized counts of the top regulators identified with iRegulon. Statistical significance was assessed using the Benjamini–Hochberg false discovery rate (FDR) <0.05 (* p < 0.05, ** p < 0.01 and *** p < 0.001). ( H , I ) Regulatory networks for the upregulated ( H ) and downregulated ( I ) genes found in MC38-FUT9 cells reveal a strong overlap among the regulons and the predicted TFs. Direct targets (DEGs) are in grey circle nodes and TFs in white circle nodes. Regulons for each TF are represented by the different line colors. # indicates the gRNA number.

Article Snippet: Cell suspensions were stained for 30 min on ice with anti-Lewis x (1:40, Merck, Kenilworth, NJ, USA, 434,631), anti-Sox2 (1:100; GeneTex, Irvine, CA, USA, GTX101507), anti-Oct4 (1:200; GeneTex, GTX100622), anti-Nanog (1:200; GeneTex, GTX627421), anti-Lewis y (1:20, GeneTex, GTX23359), anti-sialyl Lewis x (1:100, BD Pharmingen, Franklin Lakes, NJ, USA, 551344), anti-VIM2 (1:40, LabNed, Amstelveen, the Netherlands, LN1302156), isotype-APC (1:200; ImmunoTools, Friesoythe, Germany), anti-CD44-APC (1:200; ImmunoTools, 21,850,446 × 2), anti-LGR5-PE (1:100; Biolegend, 373,803), anti-CD133-BV421 (1:50; Biolegend, 372,808) and anti-CD24-AF647 (1:50; Biolegend, 311,110).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Activation Assay, CRISPR, Western Blot, Immunofluorescence, Staining, RNA Sequencing Assay

Journal: Cancers

Article Title: FUT9-Driven Programming of Colon Cancer Cells towards a Stem Cell-Like State

doi: 10.3390/cancers12092580

Figure Lengend Snippet: MC38-FUT9 cells display functional properties of cancer stem-like cells. ( A ) Representative images (left) and quantification (right) of tumorspheres formed by MC38 cells during 3D culture. Error bars represent SD. Data are representative of three independent experiments. Statistical significance was determined by an unpaired Student’s t test (*** p < 0.001). Scale bar 100 μm. ( B ) Viability of MC38-glycovariants upon treatment with indicated concentrations of 5-fluoruracil (5-FU) in 3D culture for 7 days. The percentage of living cells was determined using the CTB fluorometric assay and values were normalized to the corresponding concentration DMSO treatment for each cell line. Error bars represent SD; n = 3. Statistical significance was determined by multiple Student’s t tests (*** p < 0.001). ( C ) Cancer stem cell (CSC)-enrichment assay using flow cytometric analysis of the Lewis x antigen expression on the surface of MC38 cells cultured in 3D either alone (right panel; MC38-MOCK and MC38-FUT9) or in a heterogeneous cell suspension (left panel; FUT9-High Mix and FUT9-Low Mix) in the presence or absence of 5-FU treatment. ( D – F ) MC38-MOCK and MC38-FUT9 cells were injected under the skin (left and right flank, respectively) of NSG mice (10 mice per group) at different absolute numbers, 10 5 ( D ), 10 4 ( E ) and 10 3 ( F ), and tumor growth was monitored over time (left panel). The tumor volume during the last measurement (middle panel) and the weight of the isolated tumors (right panel) at day 17, 27 or 34 are depicted. Measurements correspond to the mice that developed both a MC38-MOCK and MC38-FUT9 tumor. Statistical differences were determined by an unpaired Student’s t test (* p < 0.05, ** p < 0.01 and *** p < 0.001).

Article Snippet: Cell suspensions were stained for 30 min on ice with anti-Lewis x (1:40, Merck, Kenilworth, NJ, USA, 434,631), anti-Sox2 (1:100; GeneTex, Irvine, CA, USA, GTX101507), anti-Oct4 (1:200; GeneTex, GTX100622), anti-Nanog (1:200; GeneTex, GTX627421), anti-Lewis y (1:20, GeneTex, GTX23359), anti-sialyl Lewis x (1:100, BD Pharmingen, Franklin Lakes, NJ, USA, 551344), anti-VIM2 (1:40, LabNed, Amstelveen, the Netherlands, LN1302156), isotype-APC (1:200; ImmunoTools, Friesoythe, Germany), anti-CD44-APC (1:200; ImmunoTools, 21,850,446 × 2), anti-LGR5-PE (1:100; Biolegend, 373,803), anti-CD133-BV421 (1:50; Biolegend, 372,808) and anti-CD24-AF647 (1:50; Biolegend, 311,110).

Techniques: Functional Assay, Concentration Assay, Expressing, Cell Culture, Injection, Isolation

Journal: Cancers

Article Title: FUT9-Driven Programming of Colon Cancer Cells towards a Stem Cell-Like State

doi: 10.3390/cancers12092580

Figure Lengend Snippet: FUT9 expression in human colon cancer cell lines is correlated with high levels of Sox2 expression and ALDH activity. ( A ) Classification of human colon cancer cell lines based on FUT9 gene expression levels. Data were obtained from the Cancer Cell Line Encyclopedia (CCLE) database. The corresponding molecular subtype for each cell line (if known) is provided . ( B ) Correlation between FUT9 and Sox2 gene expression in the subgroup of FUT9-High human colon cancer cell lines. ( C ) Gene Ontology Analysis in FUT9-High human colon cancer cells depicting all the statistically significant ( p < 0.05) groups. ( D ) Relative surface expression (MFI) of type II Lewis antigens (Lewis x , Lewis y , sialyl Lewis x and VIM2) on the surface of selected FUT9-High/Medium/Low human colon cancer cell lines, measured by flow cytometry. MFI values were normalized to the binding of the secondary antibody alone. ( E ) Cell proliferation of KM12 and HCT116 cells cultured in 2D at different absolute numbers as determined by the CTB fluorometric assay. Error bars represent SD; n = 3. Statistical differences were determined by an unpaired Student’s t test (ns; no significance). ( F ) Flow cytometric analysis of intracellular Sox2 expression in KM12 and HCT116 cells cultured in 2D. Dotted lines represent staining with the secondary antibody alone, whereas solid lines represent Sox2 staining. ( G ) Representative dotplots (left) and quantification (right) of ALDH activity in KM12 and HCT116 cells cultured in 2D, measured by the ALDEFLUOR assay. The DEAB inhibitor of ALDH was used as a control for gating and further analysis of the percentage of ALDH-High cells for each condition ( n = 4). Statistical differences were determined by an unpaired Student’s t test (** p < 0.01). ( H ) Flow cytometric analysis of Lewis x expression on the surface of KM12 cells glyco-engineered with CRISPR-Cas9. Grey lines represent staining with the second antibody only, red lines represent Lewis x staining. Numbers in red represent the MFI values for Lewis x staining normalized to the binding of the second antibody only. Histograms representative of two independent experiments are shown. ( I ) Representative dotplots of ALDH activity in KM12-MOCK and KM12-FUT9 KO cells measured by the ALDEFLUOR assay. The DEAB inhibitor of ALDH was used as a control for gating and the percentage of ALDH-High cells was calculated for each cell line.

Article Snippet: Cell suspensions were stained for 30 min on ice with anti-Lewis x (1:40, Merck, Kenilworth, NJ, USA, 434,631), anti-Sox2 (1:100; GeneTex, Irvine, CA, USA, GTX101507), anti-Oct4 (1:200; GeneTex, GTX100622), anti-Nanog (1:200; GeneTex, GTX627421), anti-Lewis y (1:20, GeneTex, GTX23359), anti-sialyl Lewis x (1:100, BD Pharmingen, Franklin Lakes, NJ, USA, 551344), anti-VIM2 (1:40, LabNed, Amstelveen, the Netherlands, LN1302156), isotype-APC (1:200; ImmunoTools, Friesoythe, Germany), anti-CD44-APC (1:200; ImmunoTools, 21,850,446 × 2), anti-LGR5-PE (1:100; Biolegend, 373,803), anti-CD133-BV421 (1:50; Biolegend, 372,808) and anti-CD24-AF647 (1:50; Biolegend, 311,110).

Techniques: Expressing, Activity Assay, Flow Cytometry, Binding Assay, Cell Culture, Staining, CRISPR

Journal: Frontiers in Oncology

Article Title: Differential O - and Glycosphingolipid Glycosylation in Human Pancreatic Adenocarcinoma Cells With Opposite Morphology and Metastatic Behavior

doi: 10.3389/fonc.2020.00732

Figure Lengend Snippet: Immune recognition of glycan structures on PaTu-S and PaTu-T cells. (A) Interaction of immature DCs with PaTu-S and PaTu-T were visualized by fluorescence microscopy. Bar = 100 μm. (B) Binding of immature DCs to PaTu-S and PaTu-T in a cell adhesion assay, in the presence or absence of EGTA. Results are derived from 6 independent experiments using different donors and expressed as average percentage binding ± SEM. (C) Binding of recombinant human galectins Gal-1, Gal-3, and Gal-4 (5 μg/ml) to the PDAC cell lines was measured by flow cytometry. Results are given as average MFI ± SEM of at least 2 independent experiments. (D) Binding of Fc-chimeras of DC-SIGN, MGL, DCIR and Dectin-1 to PaTu-S and PaTu-T cells was measured by flow cytometry. Results are given as average MFI ± SEM of at least 3 independent experiments. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: Mouse anti-human galectin (Gal)-1 , and anti-Tn monoclonal antibodies were kindly provided by Dr. RD Cummings (Boston, USA), goat anti-human Gal-4 was purchased from R&D Systems (Minneapolis, MN) and anti-sialyl Lewis A/CA 19-9 monoclonal antibody was from LifeSpan Biosciences (Seattle, WA).

Techniques: Fluorescence, Microscopy, Binding Assay, Cell Adhesion Assay, Derivative Assay, Recombinant, Flow Cytometry