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

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: Fibroblast-secreted galectin-1 (Gal-1) significantly promotes multiple CIC features in CRC cells. (A) Expression of endogenous Gal-1 in MRC-5 and WS1 fibroblasts as detected through Western blot (top panel; recombinant human Gal-1 protein (rhGal-1) used as positive control) and ELISA (bottom panel). (B) Invasion capacity of KM12C with the addition of increasing doses of rhGal-1. (C) qPCR analysis for the gene expression of Twist1 and E-cadherin and (D) Western blot for protein expression of Slug and E-cadherin in KM12C with the addition of increasing doses of rhGal-1. (E) IF staining of E-cadherin (green fluorescence) in KM12C with the addition of increasing doses of rhGal-1 for 48 hours. Nuclei were stained with Hoechst 33342 (blue fluorescence); scale bar, 10 μm. (F) Sphere formation capacity of KM12C with the addition of increasing doses of rhGal-1; quantitative results (top panel) and representative images (bottom panel) are shown; scale bar, 30 μm. (G) Drug resistance capacity of KM12C to cisplatin (25 µM) after pretreatment with increasing dosages of rhGal-1 for 24 hours. Cell viability was assessed 48 hours after drug treatment. (H) Left panel: Validation of Gal-1 knockdown using small interfering RNA (siRNA) specific for Gal-1 (siRNA-I & siRNA-II) in WS1 fibroblasts. Non-target siRNA was used as a negative control. After 48 hours, CM from siRNA-I, siRNA-II, and siC were removed and analyzed by ELISA. Right panel: Invasion capacity of KM12C after culturing in siGal-WS1-CM compared to siC-WS1-CM for 48 hours. (I) Sphere formation capacity of KM12C cultured in KM12C-CM, siC-WS1-CM, or siGal-WS1-CM for 72 hours; quantitative results (top panel) and representative images (bottom panel) are shown; scale bar, 30 μm. (J) Drug resistance capacity of KM12C to cisplatin (25 µM) after pretreatment with KM12C-CM, siGal-WS1-CM, or siC-WS1-CM for 24 hours. Cell viability was assessed 48 hours after drug treatment; control, KM12C without cisplatin treatment. All results are shown as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01, and *** p < 0.005 compared to the control.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: Expressing, Western Blot, Recombinant, Positive Control, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Small Interfering RNA, Negative Control, Cell Culture

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: Fibroblast-secreted Gal-1 significantly increases metastasis and tumor dissemination of CRC cells in vivo . (A) Body weight of NOD-SCID mice 40 days after tail vein injection of KM12C only (3 x 10 5 cells); KM12C (3 x 10 5 cells) admixed with WS1 silenced for with short-hairpin RNA (shRNA) of non-target sequences (shC-WS1; 3 x 10 5 cells); KM12C (3 x 10 5 cells) admixed with WS1 silenced with shRNA specific for Gal-1 (shGal-WS1; 3 x 10 5 cells); or shC-WS1 only (3 x 10 5 cells). Each condition consisted of three mice, with their body weight measured every 7 days. Immunohistochemistry (IHC) staining for human histone H1 (brown nuclei) in (B) mouse lung and (C) spleen tissue sections; representative sections (top panel) and quantitative results (bottom panel) are shown, with arrows indicating human Histone H1(+) cells; scale bar, 20 μm. (D) Visualization of fluorescently labeled co-cultured cells KM12C (3 x 10 5 cells; green fluorescence, labeled with DiO), and siC- or siGal-WS1 (3 x 10 5 cells; red fluorescence, labeled with with DiI) in lung sections 24 hours after injection into the tail vein of C57BL/6 mice. Top panel, representative images; bottom panel; quantitative results. Arrows indicate KM12C; scale bar, 100 μm. All results are shown as the mean ± SEM of three independent experiments. * p < 0.05, and *** p < 0.005 compared to the control. (E) Analyses of Gal-1 ( LGALS1 ) and β-catenin ( CTNNB1 ) expression with regard to disease recurrence in the public dataset GSE33113 and GSE17536 of gene expression omnibus (GEO); * p < 0.05; N.S., not significant.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: In Vivo, Injection, shRNA, Immunohistochemistry, Labeling, Cell Culture, Fluorescence, Expressing

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: Gal-1 promotes β-catenin expression, nuclear translocation, and activity in CRC cells. (A) IF staining for β-catenin (green fluorescence) in KM12C with the addition of increasing doses of rhGal-1 for 48 hours. Nuclei were stained with Hoechst 33342 (blue fluorescence). Arrows show nuclear β-catenin; scale bar, 10 μm. (B) Western blot for β-catenin levels in whole cell lysate (top panel) and nuclear fraction (bottom panel) of KM12C with the addition of increasing doses of rhGal-1 for 48 hours; for nuclear fraction, histone H1 is used as the positive control and α-Tubulin as the negative control. (C) Luciferase reporter assay for β-catenin activity in KM12C with the addition of increasing doses of rhGal-1. TOPFlash plasmids (β-catenin promoter reporter construct containing TCF/LEF binding sites; please see Materials and Methods) and TOPFlash mutant plasmids (β-catenin promoter reporter construct containing mutated TCF/LEF binding sites; please see Materials and Methods) were transduced into KM12C, with the luciferase activity measured 48 hours later; addition of the Wnt/β-catenin agonist CHIR-99021 (CHIR; 0.3 µM) was used as a positive control. (D) qPCR analysis for the gene expression of Twist1 in KM12C after treatment with rhGal-1 (100 ng/ml) and without or with the Wnt/β-catenin antagonist XAV-939 (XAV; 10 µM) for 24 hours. All results are shown as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01, and *** p < 0.005 compared to the control.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: Expressing, Translocation Assay, Activity Assay, Staining, Fluorescence, Western Blot, Positive Control, Negative Control, Luciferase, Reporter Assay, Construct, Binding Assay, Mutagenesis

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: SOX9 is a critical mediator involved in Gal-1-induced upregulation of β-catenin activity and CIC features. (A) Ingenuity Pathway Analysis (IPA) for the prediction of candidate mediators within the LGALS1/CTNNB1/Twist1 axis. IPA database revealed the several major pathways which might be involved in tumor development and metastasis. According to the IPA results and literature review, SOX9 was selected and confirmed whether it is the downstream gene of Gal-1 by Western blot. (B) Western blot for the analysis of SOX9 protein levels in KM12C after culturing in MRC-5- or WS1-CM; KM12C-CM was used as the control. Internal control: GAPDH. (C) Western blot for SOX9 levels in whole cell lysate (top panel) and nuclear fraction (bottom panel) of KM12C with addition of increasing doses of rhGal-1 for 48 hours; for nuclear protein blot, histone H1 is used as the positive control and α-Tubulin as the negative control. (D) Sphere formation capacity of shC-KM and shSOX9-II-KMC12 (shSOX9-KM) after treating with rhGal-1 (100 ng/ml) for 72 hours. (E) Drug resistance capacity of shC- and shSOX9-KM to cisplatin (25 µM) after pretreatment with rhGal-1 (100 ng/ml) for 24 hours. Cell viability was assessed 48 hours after drug treatment. (F) qPCR analysis for the gene expression of Twist1 in shC- and shSOX9-KM after treatment with Gal-1 (100 ng/ml) and XAV (10 µM). (G) Invasion capacity of shC- and shSOX9-KM with addition of rhGal-1 (100 ng/ml) and XAV (10 µM). All results are shown as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01, and *** p < 0.005 compared to the control. N.S., not significant.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: Activity Assay, Western Blot, Positive Control, Negative Control, Expressing

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: High expression of Gal-1 and SOX9 correlate with clinical CRC outcome. (A) ONCOMINE assessment of the expression levels of LGALS1 and SOX9 in the Kaiser Colon database. (B) Analysis of LGALS1 or SOX9 expression levels in tumor tissue compared to adjacent normal tissue using GSE9348 (top panel) and The Cancer Genome Atlas (TCGA) databases (bottom panel). * p < 0.05 and *** p < 0.005 for early-stage lesions compared to adjacent normal tissue. (C) Analysis of LGALS1 and SOX9 expression levels to the CRC stage using the GSE17536 and TCGA datasets. (D) Immunohistological staining of Gal-1 and SOX9 on CRC tumor samples, which included 40 primary lesions (primary), 10 metastatic lesions (metastatic), and 9 normal colon samples; scale bar, 100 μm. (E) Kaplan-Meier survival curves of four groups of CRC patients as stratified by median expression levels of SOX9 and Gal-1 in tumor tissue: SOX9 low /Gal-1 low , n = 38; SOX9 low /Gal-1 high & SOX9 high /Gal-1 low , n = 100; and SOX9 high /Gal-1 high , n = 39. Survival analyses was performed for two groups: SOX9 high /Gal-1 high versus SOX9 low /Gal-1 low (left-side graph); or for three groups: SOX9 high /Gal-1 high versus SOX9 high /Gal-1 low + SOX9 low /Gal-1 high versus SOX9 low /Gal-1 low (right-side graph).

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: Expressing, Staining

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: Direct targeting of stromal-secreted Gal-1 on CRC cells promote CIC features and disease progression through SOX9 and β-catenin.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques:

Journal: Scientific Reports

Article Title: Ferroquine, the next generation antimalarial drug, has antitumor activity

doi: 10.1038/s41598-017-16154-2

Figure Lengend Snippet: Ferroquine arrests autophagy. ( A ) Transmission electron microscopy images of LNCaP cells treated with vehicle, CQ (10 μM) or FQ (10 μM) for 24 h. Scale bar, 1 μm. (B) Immunoblotting for LC3, p62 and Actin as a loading control. LNCaP cells were treated with CQ or FQ for 12 h. ( C) Representative confocal images of LNCaP cells transfected with eGFP-LC3 and treated as in ( A ). Scale bars, 10 μm. (D) Quantification of ( C ) (n = 100). Mean ± SEM; Mann-Whitney test; ***P < 0.001. (E) Effect of FQ (20 μM), ferrocene (20 μM) and ferrocene + CQ (20 μM) on autophagy in LNCaP cells. Immunoblotting for LC3 and Actin. (F) Immunoblotting for LC3 and Actin. LNCaP cells were treated with vehicle, CQ (10 μM) or FQ (10 μM) for 6 h in complete medium or in glucose-free salt solution. Full-length blots are included in the Supplementary Information.

Article Snippet: Mouse anti-p62 (D-3, sc-28359), mouse anti-Lamp1 (H4A3, sc-20011), mouse anti-HIF-1α (28b, sc-13515), rabbit anti-Cathepsin B (sc-6493), mouse anti-Galectin 3 (sc-32790), DL-α-Tocopherol (sc-294383A), H-Leu-Leu-OMe Hydrochloride (sc-285992A), Trolox (sc-200810), Ferrocene (sc-353607), Deferoxamine mesylate (sc-203331) were from Santa Cruz Biotechnology.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Control, Transfection, MANN-WHITNEY