Journal: Viruses

Article Title: Oncolytic H-1 Parvovirus Hijacks Galectin-1 to Enter Cancer Cells

doi: 10.3390/v14051018

Figure Lengend Snippet: H-1PV transduction is reduced in LGALS1 , but not LGALS3 , knockdown cell lines. HeLa, NCH125 and BxPC3 cells were transfected with siRNAs targeting LGALS1 or LGALS3 or with a scrambled siRNA. At 48 h post-transfection, cells were infected with recH-1PV-EGFP for 4 h and grown for an additional 20 h. Cells were then processed as described in the Materials and Methods. Numbers represent the arithmetic mean percentage of EGFP-positive cells relative to the number of EGFP-positive cells observed in cells transfected with control siRNA, which was arbitrarily set as 100%. The independent experiment shown was repeated thrice each with three biologically independent samples ( ns–not significant ; *** p ≤ 0.001, calculated by using a one-way ANOVA).

Article Snippet: To rescue LGALS1 expression, the plasmid encoding LGALS1 gene was used (SC118705; OriGene Technologies, Inc. Rockville, MD, USA).

Techniques: Transduction, Transfection, Infection

Journal: Viruses

Article Title: Oncolytic H-1 Parvovirus Hijacks Galectin-1 to Enter Cancer Cells

doi: 10.3390/v14051018

Figure Lengend Snippet: H-1PV infectivity is reduced in NCH125 LGALS1 KO cells. ( A ) H-1PV entry decreases in NCH125 LGALS1 KO cells. Control and LGALS1 KO cells were infected with H-1PV at an MOI of 50 pfu/cell for 2 h at 37 °C and prepared for confocal microscopy analysis. Gal-1 (green) and H-1PV capsid (red) were detected using specific antibodies, while DAPI (blue) was used to stain nuclei. As expected, Gal-1 was readily detected in Control cells, while it fell below detection limits in LGALS1 KO cells. The lower panel shows representative examples of H-1PV-infected cells. Quantification of the H-1PV fluorescence signal is shown on the right. This was retrieved from two independent experiments in which the fluorescence intensity was quantified in 25 randomly identified cells using ImageJ. Box plot depicts the median with a centre line, and the Tukey–Whiskers plots indicate variability outside the upper and lower quartiles ( n = 25, *** p ≤ 0.001). ( B ) Control and LGALS1 KO cells were infected with H-1PV at an MOI of 2 pfu/cell for 48 h, and NS1 and VP1 protein levels were assessed by Western blotting. Beta-tubulin was used as a loading control. ( C ) H-1PV transduction is decreased in LGALS1 KO cells and re-established by transfecting the cells with a plasmid carrying LGALS1 . LGALS1 KO cells were transfected with a plasmid encoding LGALS1 , treated only with lipofectamine LTX (mock transfection) or left untreated. In addition, 48 h post-transfection, cells were infected with recH-1PV-EGFP for 24 h. Control cells were also included, and the level of virus transduction was set arbitrarily at 100%. The independent experiment shown was repeated twice each with four biologically independent samples ( n s : p > 0.05; *** p ≤ 0.001, calculating using a one-way ANOVA). On the right side, Western blotting analysis shows the levels of Gal-1 at the time of infection. Βeta-tubulin was used as a loading control.

Article Snippet: To rescue LGALS1 expression, the plasmid encoding LGALS1 gene was used (SC118705; OriGene Technologies, Inc. Rockville, MD, USA).

Techniques: Infection, Confocal Microscopy, Staining, Fluorescence, Western Blot, Transduction, Plasmid Preparation, Transfection

Journal: Viruses

Article Title: Oncolytic H-1 Parvovirus Hijacks Galectin-1 to Enter Cancer Cells

doi: 10.3390/v14051018

Figure Lengend Snippet: H-1PV has reduced oncolytic activity in NCH125 LGALS1 KO cells, which is rescued by supplementing with recombinant Gal-1 protein; ( A ) H-1PV oncolytic activity is reduced in NCH125 LGALS1 KO cells. Control and LGASL1 KO cells were infected with H-1PV at an MOI of 1 pfu/cell. Cell viability was assessed by MTT every 24 h for a total of 96 h. The curve plot depicts the mean ± standard deviation for each time point expressed as a percentage of cell viability compared to corresponding uninfected cells. The independent experiment shown was repeated thrice each with four biologically independent samples (*** p ≤ 0.001). ( B ) Purified Gal-1 rescues H-1PV oncolytic activity in NCH125 LGALS1 KO cells. Control and LGASL1 KO cells were infected (or not) with H-1PV at an MOI of 1 pfu/cell, in the presence or absence of 5 μg/mL of human recombinant Gal-1. Cell viability was assessed at 72 h post-infection by MTT. Columns depict the percentage (mean value) of cell viability compared to uninfected cells ± standard deviation bars. The independent experiment shown was repeated twice each with four biologically independent samples ( ns: p > 0.05; *** p ≤ 0.001, calculated using a one-way ANOVA).

Article Snippet: To rescue LGALS1 expression, the plasmid encoding LGALS1 gene was used (SC118705; OriGene Technologies, Inc. Rockville, MD, USA).

Techniques: Activity Assay, Recombinant, Infection, Standard Deviation, Purification

Journal: Viruses

Article Title: Oncolytic H-1 Parvovirus Hijacks Galectin-1 to Enter Cancer Cells

doi: 10.3390/v14051018

Figure Lengend Snippet: H-1PV cell entry, but not cell attachment, is reduced in NCH125 LGALS1 KO cells. ( A ) H-1PV binding and entry assay assessed by qPCR. NCH125 Control and LGALS1 KO cells were infected with H-1PV at an MOI of 5 pfu/cell for 0.5, 1, 1.5, 2 and 4 h at 37 °C. Cells were then extensively washed and harvested, and encapsidated viral DNA was extracted and subjected to qPCR. Columns in the graph show the number of copies of the cell-associated H-1PV genome with relative standard deviations (*** p ≤ 0.001). The independent experiment shown was repeated thrice; n = 3 biologically independent samples. ( B ) Binding and entry is rescued by the addition of purified recombinant Gal-1. At the time of H-1PV infection (at an MOI of 5 pfu/cell), Gal-1 was added (or not) to the culture medium. Infection was carried out for 4 h. Numbers indicate the percentage of cell-associated genomes relative to NCH125 Control cells infected with H-1PV arbitrarily set as 100% The independent experiment shown was repeated twice each with four biologically independent samples ( ns: p > 0.05; *** p ≤ 0.001, calculated using a one-way ANOVA). ( C ) H-1PV cell surface binding assessed by Flow cytometry. A representative flow cytometry histogram with overlay of Control (black) and LGALS1 KO cells (blue) shows no difference in H-1PV-associated cells. Cells were either mock- or H-1PV-infected (at an MOI of 25 pfu/cell) for 1 h at 4 °C. Cells were not permeabilised for the Flow cytometry analysis, and cell surface-bound H-1PV particles were detected with a specific anti-capsid antibody. The independent experiment shown was repeated twice each with two biologically independent samples. ( D ) H-1PV binding only, assessed by qPCR. Control and LGALS1 KO cells were infected with H-1PV (at an MOI of 5 pfu/cell) for 1 h at 4 °C. Cells were then washed and harvested, and extracted encapsidated viral DNA was quantified by qPCR. The independent experiment shown was repeated thrice each with three biologically independent samples.

Article Snippet: To rescue LGALS1 expression, the plasmid encoding LGALS1 gene was used (SC118705; OriGene Technologies, Inc. Rockville, MD, USA).

Techniques: Cell Attachment Assay, Binding Assay, Infection, Purification, Recombinant, Flow Cytometry

Journal: Viruses

Article Title: Oncolytic H-1 Parvovirus Hijacks Galectin-1 to Enter Cancer Cells

doi: 10.3390/v14051018

Figure Lengend Snippet: Effect on H-1PV binding and entry upon depletion of both LAMC1 and LGALS1 in NCH125 and HeLa cells. ( A ) NCH125 Control and LGALS1 KO cells were transfected with a siRNA targeting LAMC1 or a negative control siRNA. At 48 h post-transfection, cells were infected with H-1PV at an MOI of 5 pfu/cell for 4 h at 37 °C. Cells were then extensively washed and harvested, and encapsidated viral DNA was extracted and subjected to qPCR. Columns in the graph show the number of copies of the cell-associated H-1PV genome with relative standard deviations ( ns : p > 0.05; *** p ≤ 0.001, calculated using a one-way ANOVA). The independent experiment shown was repeated thrice each with threebiologically independent samples. ( B ) HeLa Control and HeLa LAMC1 KD cells were transfected with a siRNA targeting LGALS1 or a negative control siRNA. After 48 h siRNA transfection, cells were infected with H-1PV at an MOI of 5 pfu/cell for 4 h at 37 °C. Cells were then processed as per A . The experiment was performed with four biologically independent samples (** p ≤ 0.01, calculated using a one-way ANOVA).

Article Snippet: To rescue LGALS1 expression, the plasmid encoding LGALS1 gene was used (SC118705; OriGene Technologies, Inc. Rockville, MD, USA).

Techniques: Binding Assay, Transfection, Negative Control, Infection

Journal: Viruses

Article Title: Oncolytic H-1 Parvovirus Hijacks Galectin-1 to Enter Cancer Cells

doi: 10.3390/v14051018

Figure Lengend Snippet: Differential expression of Gal-1 in normal tissues and in primary and recurrent GBM biopsies. ( A ) overview of the tissue microarray. This study included biopsies from normal tissue ( n = 12), primary GBM biopsies ( n = 61) and recurrent GBM biopsies ( n = 49). Biopsies were categorised based on Gal-1 expression after immunostaining with anti-galectin-1 antibody: low (<10% positive cells), medium (10–30% positive cells) or high expression (>30% positive cells). The number of biopsies in each category are indicated under each representative image. The staining was performed twice in each normal sample and thrice on tumour tissues. Quantification of Gal-1-positive cells (%) was performed as described in the Materials & Methods section using in-house software; ( B ) comparative analysis of Gal-1 expression between healthy tissues and GBM biopsies (primary and recurrent); ( C ) comparative analysis of Gal-1 expression between primary and recurrent GBM biopsies. The arithmetic mean of Gal-1-positive cells is indicated with a horizontal line and by the number above (** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: To rescue LGALS1 expression, the plasmid encoding LGALS1 gene was used (SC118705; OriGene Technologies, Inc. Rockville, MD, USA).

Techniques: Expressing, Microarray, Immunostaining, Staining, Software

Journal: Viruses

Article Title: Oncolytic H-1 Parvovirus Hijacks Galectin-1 to Enter Cancer Cells

doi: 10.3390/v14051018

Figure Lengend Snippet: Correlation between LGALS1 gene expression of cancer cell lines and their susceptibility to H-1PV-induced oncolysis. ( A ) LGALS1 gene expression was retrieved from the National Cancer Institute (NCI)-60 database. Fifty-three cancer cell lines from the NCI-60 panel were tested for their susceptibility to H-1PV infection by xCELLigence. H-1PV EC50 values were calculated as the viral MOI that kills 50% of the cell population at 72 h post-infection (72hpi), measured by xCELLigence (see also ). Six cancer cell lines (MCF7, COLO 205, HCC-2998, HCT-15, LOX IMVI, OVCAR-3 (indicated by arrows) were found to be resistant to cell lysis even at the maximum tested concentrations of H-1PV (MOI 50 pfu/cell). Therefore, as EC50 values could not be calculated for those cell lines, their values were arbitrarily fixed as 100; ( B ) LGALS1 expression versus H-1PV EC50. Each blue dot corresponds to a cell line and the grey line corresponds to a linear regression. ( C ) LGALS1 levels are moderately anti-correlated with H-1PV EC50. LGALS1 gene expression measurements were retrieved from the NCI-60 (53 cell lines) and Cancer Cell Line Encyclopedia (CCLE) (52 cell lines). Bar plot depicts the correlation between the gene expression from each dataset and the EC50 values (Pearson’s correlation). Significant anti-correlation was observed for both NCI-60 and CCLE datasets with R = –0.510, C.I. (–0.685, –0.277), p < 0.001 and –0.422, C.I. (–0.641, –0.139), p < 0.01 respectively (in both cases, the null hypothesis: R = 0).

Article Snippet: To rescue LGALS1 expression, the plasmid encoding LGALS1 gene was used (SC118705; OriGene Technologies, Inc. Rockville, MD, USA).

Techniques: Expressing, Infection, Lysis

Journal: Viruses

Article Title: Oncolytic H-1 Parvovirus Hijacks Galectin-1 to Enter Cancer Cells

doi: 10.3390/v14051018

Figure Lengend Snippet: Galectin-1 levels in glioma cell lines determine the success of H-1PV infection. ( A ) Total mRNA was isolated from glioma cell lines susceptible (NCH125; NCH37) or semi-permissive (U251; LN308; T98G; A172-MG) to H-1PV infection, and LGALS1 mRNA transcripts were measured using nCounter analysis. Bar graph depicts the LGALS1 transcript counts; numbers on the top of the columns indicate gene expression fold changes between susceptible and semi-permissive cancer cell lines. The independent experiment is shown; n = 1 (NCH125, NCH37, U251 and A172-MG); n = 2 (LN308 and T98G); n = 3 (human astrocytes) biologically independent samples. ( B ) NCH125 and NCH37 cell lines were either infected with H-1PV at an MOI of 5 pfu/cell (green) or left untreated (red). Semi-permissive cell lines were infected with H-1PV at an MOI of 5 pfu/cell (green), incubated with 5 μg/mL of human recombinant Gal-1 (pink), H-1PV and Gal-1 simultaneously (blue), or left untreated (red). Cell viability was assessed by xCELLigence every 30 min in real time. The curve shows the “Cell index” mean of three biologically independent samples ( n = 3) at any given time, which is proportional to the viability of the cell population. Black arrows indicate the time of treatment. ( C ) H-1PV binding at the cell surface is not affected by Gal-1 addition. U251, LN308, T98G and A172-MG cells were incubated with H-1PV alone at an MOI of 5 pfu/cell or with H-1PV and Gal-1. Incubations were carried out for 1 h at 4 °C (binding only). Cells were then washed and harvested, and encapsidated viral DNA was extracted and subsequently quantified by qPCR. Columns in the graph show the fold change of number of copies of cell-associated H-1PV genome relative to the virus-infected cells arbitrarily set as 1, with respective standard deviations. The independent experiment shown was performed with four biologically independent samples ( ns : p > 0.05; *** p ≤ 0.001, calculated using a one-way ANOVA). ( D ) H-1PV entry is rescued upon Gal-1 addition. U251, LN308, T98G and A172-MG cells were incubated with H-1PV alone at an MOI of 5 pfu/cell or with H-1PV and Gal-1. Incubations were carried out either for 4 h at 37 °C (binding and entry). Cells were processed and results analysed as described in C.

Article Snippet: To rescue LGALS1 expression, the plasmid encoding LGALS1 gene was used (SC118705; OriGene Technologies, Inc. Rockville, MD, USA).

Techniques: Infection, Isolation, Expressing, Incubation, Recombinant, Binding Assay